Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids

ABSTRACT

The present invention relates to the fields of medicine, cell biology, molecular biology and genetics. In particular, the present invention provides methods to isolate and purify microvesicles from cell culture supernatants and biological fluids. The present invention also provides pharmaceutical compositions of microvesicles to promote or enhance wound healing, stimulate tissue regeneration, remodel scarred tissue, modulate immune reactions, alter neoplastic cell growth and/or mobility, or alter normal cell growth and/or mobility. The present invention also provides compositions of microvesicles to be used as diagnostic reagents, and methods to prepare the compositions of microvesicles.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/775,382, filed Sep. 11, 2015, which is a 35 U.S.C. § 371 filing ofInternational Patent Application No. PCT/US2014/024629, filed Mar. 12,2014, which claims priority to U.S. Patent Application Ser. No.61/778,591, filed Mar. 13, 2013, the entire disclosures of which arehereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine, cell biology,molecular biology and genetics. In particular, the present inventionprovides methods to isolate and purify microvesicles from cell culturesupernatants and biological fluids. The present invention also providespharmaceutical compositions of microvesicles to promote or enhance woundhealing, stimulate tissue regeneration, remodel scarred tissue, modulateimmune reactions, alter neoplastic cell growth and/or mobility, or alternormal cell growth and/or mobility. The present invention also providescompositions of microvesicles to be used as diagnostic reagents, andmethods to prepare the compositions of microvesicles.

BACKGROUND

Microvesicles are secreted by many, if not all, cell types in vitro andin vivo, and are present in biological fluids, such as, for example,blood, interstitial fluid, urine, saliva, and tears. Microvesicles arevesicles comprising lipid bilayers, formed from the plasma membrane ofcells, and are heterogeneous in size, ranging from about 2 nm to about5000 nm. The cell from which a microvesicle is formed is herein referredto as “the host cell”. Microvesicles are a heterogeneous population ofvesicles and include, for example, ectosomes, microparticles,microvesicles, nanovesicles, shedding vesicles and membrane particles.

Microvesicles exhibit membrane proteins from their host cell on theirmembrane surface, and may also contain molecules within the microvesiclefrom the host cell, such as, for example, mRNA, miRNA, tRNA, RNA, DNA,lipids, proteins or infectious particles. These molecules may resultfrom, or be, recombinant molecules introduced into the host cell.Microvesicles play a critical role in intercellular communication, andcan act locally and distally within the body, inducing changes in cellsby fusing with a target cell, introducing the molecules transported onand/or in the microvesicle to the target cell. For example,microvesicles have been implicated in anti-tumor reversal, cancer, tumorimmune suppression, metastasis, tumor-stroma interactions, angiogenesisand tissue regeneration. Microvesicles may also be used to diagnosedisease, as they have been shown to carry bio-markers of severaldiseases, including, for example, cardiac disease, HIV and leukemia.

Despite the importance of microvesicles, isolating microvesicles inuseful quantities, while preserving their structural and functionalintegrity, remains problematic. The traditional procedure utilizesultracentrifugation to isolate microvesicles from samples.

For example, U.S. Pat. No. 7,807,438 discloses a method for isolation ofhepatitis C virus. The method comprises the separation of particlestermed exosomes from the blood plasma of an individual infected withhepatitis C virus (HCV) and the extraction of RNA from these exosomeparticles.

In another example, U.S. Patent Application US20030198642A1 discloses[e]xosomes . . . derived from MHC class II enriched compartments inantigen presenting cells . . . [for] a . . . vaccination vehicle.

In another example, U.S. Patent Application US20060116321A1 disclosesmethods and compositions for use in mediating an immunosuppressivereaction. The compositions . . . comprise exosomes havingimmunosuppressive activity. Such exosomes may be derived from a varietyof different cell types, including antigen-presenting cells such asdendritic cells and macrophages. Prior to isolation of exosomes, thecells may be genetically engineered to express molecules capable ofenhancing the immunosuppressive activity of said exosomes and/or may beexposed to one or more agents, such as cytokines or cytokine inhibitors,which are also capable of enhancing the immunosuppressive activity ofexosomes. The present invention also relates to the use of such exosomesfor the treatment of diseases and disorders associated with undesirableactivation of the immune system. The present invention also includesexosomes isolated directly from serum that have been shown to beimmunosuppressive.

Ultracentrifugation may damage the microvesicles, resulting in the lysisor rupture of the vesicles. Damage to microvesicles may cause an adversereaction in the body, if such damaged microvesicles were to beintroduced.

Others have attempted alternate methods to isolate microvesicles.However, the alternate methods employed frequently isolate asub-fraction of microvesicles, or are inefficient. For example, U.S.Patent Application US2011003008A1 discloses a particle secreted by amesenchymal stem cell and comprising at least one biological property ofa mesenchymal stem cell. The biological property may comprise abiological activity of a mesenchymal stem cell conditioned medium(MSC-CM) such as cardioprotection or reduction of infarct size. Theparticle may comprise a vesicle or an exosome.

In another example, U.S. Patent Application US20120070858A1 discloses amethod for isolating exosomes from blood platelets usingsuperparamagnetic nanoparticles of iron oxide (Fe₃O₄), by means of acharge attraction mechanism based on the predetermined Zeta potential ofthe exosomes. The method involves the use of iron oxide nanoparticlesthat are previously synthesised [sic] with a predetermined positivecharge, and that bond to the negatively charged exosomes contained inthe biological sample. During incubation, the cationic magneticnanoparticles are absorbed by the surface of the membrane of theexosomes owing to electrostatic interaction. Exposure of the material toa magnetic field makes it possible to separate the exosomes bonded tothe nanoparticles. The success of this technique has been confirmed bycharacterisation [sic] of the exosomes by flow citometry [sic]. Themethod has been shown to be suitable for this purpose, since it allowsexosomes to be isolated and purified, without undergoing alterations oftheir original morphological and structural characteristics.

In another example, PCT Patent Application WO2012169970A1 disclosesmaterials and methods for use of constrained cohydration agents in thepurification of biological materials such as antibodies, viruses, cells,and cellular organelles in connection with convective chromatography,fluidized bed or co-precipitation applications.

There remains, therefore, a need to provide method to isolate and purifymicrovesicles without damage, and in sufficient quantities that theisolated microvesicles may subsequently be used for diagnosing disease,therapies, or research.

The present invention provides methods to isolate microvesicles frombiological fluids without damaging the structural and/or functionalintegrity of the microvesicles. The present invention also providesmethods to isolate ectosomes, microparticles, microvesicles,nanovesicles, shedding vesicles, apoptotic bodies, or membrane particlesfrom biological fluids without damaging their structural and/orfunctional integrity.

SUMMARY

In one embodiment, the present invention provides a method for isolatingand/or purifying microvesicles from cell culture supernatants orbiological fluids utilizing precipitation agent that precipitates themicrovesicle from the cell culture supernatant or biological fluid bydisplacing the water of solvation.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles. In one embodiment, the isolatedpreparation of microvesicles is subsequently purified. In oneembodiment, the isolated preparation of microvesicles is subsequentlypurified to yield a preparation of ectosomes. In one embodiment, theisolated preparation of microvesicles is subsequently purified to yielda preparation of microparticles. In one embodiment, the isolatedpreparation of microvesicles is subsequently purified to yield apreparation of nanovesicles. In one embodiment, the isolated preparationof microvesicles is subsequently purified to yield a preparation ofshedding vesicles. In one embodiment, the isolated preparation ofmicrovesicles is subsequently purified to yield a preparation ofmembrane particles. In one embodiment, the isolated preparation ofmicrovesicles is subsequently purified to yield a preparation ofapoptotic bodies.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that promotes or enhances angiogenesis. Inone embodiment, the isolated preparation of microvesicles promotes orenhances angiogenesis in a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that promotes or enhances neuronalregeneration. In one embodiment, the isolated preparation ofmicrovesicles promotes or enhances neuronal regeneration in a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that promotes or enhances cellularproliferation. In one embodiment, the isolated preparation ofmicrovesicles promotes or enhances cellular proliferation in a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that promotes or enhances cellularmigration. In one embodiment, the isolated preparation of microvesiclespromotes or enhances cellular migration in a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that promotes or enhances wound healing. Inone embodiment, the wound is a full-thickness burn. In one embodiment,the wound is a second-degree burn.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that reduces scar formation in a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that reduces wrinkle formation in the skinof a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that is used to diagnose the presenceand/or progression of a disease in a patient. In one embodiment, thedisease is metastatic melanoma. In an alternative embodiment the diseasein an inflammatory/autoimmune disorder such as rheumatoid arthritis. Inone embodiment, the disease is graft versus host disease.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that can promote functional regenerationand organization of complex tissue structures. In one embodiment thepresent invention provides an isolated preparation of microvesicles thatcan regenerate hematopoietic tissue in a patient with aplastic anemia.In one embodiment the present invention provides an isolated preparationof microvesicles that can regenerate at least one tissue in a patientwith diseased, damaged or missing skin selected from the groupconsisting of: epithelial tissue, stromal tissue, nerve tissue, vasculartissue and adnexal structures. In one embodiment, the present inventionprovides an isolated preparation of microvesicles that can regeneratetissue and/or cells from all three germ layers.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that is used to modulate the immune systemof a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that enhances the survival of tissue orcells that is transplanted into a patient. In one embodiment, thepatient is treated with the isolated preparation of microvesicles priorto receiving the transplanted tissue or cells. In an alternateembodiment, the patient is treated with the isolated preparation ofmicrovesicles after receiving the transplanted tissue or cells.

In an alternate embodiment, the tissue or cells is treated with theisolated preparation of microvesicles. In one embodiment, the tissue orcells is treated with the isolated preparation of microvesicles prior totransplantation.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles containing at least one molecule selectedfrom the group consisting of RNA, DNA, and protein from a host cell. Inone embodiment, the host cell is engineered to express at least onemolecule selected from the group consisting of RNA, DNA, and protein. Inone embodiment, the isolated preparation of microvesicles containing atleast one molecule selected from the group consisting of RNA, DNA, andprotein from a host cell is used as a therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention and, together with the description, further serve to explainthe principles of the invention and to enable a person of ordinary skillin the art to make and use the invention. In the drawings, likereference numbers indicate identical or functionally similar elements. Amore complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows a schematic outline of a protocol used to isolatemicrovesicles by ultracentrifugation.

FIG. 2 shows one embodiment of a microvesicle isolation method of thepresent invention.

FIG. 3 shows an alternate embodiment of a microvesicle isolation methodof the present invention.

FIG. 4 shows one embodiment of an apparatus of the present inventionthat facilitates the clarification of the biological fluid and thecollection of the precipitated microvesicles by filtration.

FIG. 5A-FIG. 5D show electron micrographs of microvesicles derived frommedium conditioned using human bone marrow-derived mesenchymal stemcells isolated by the ultracentrifuge method described in Example 1(FIG. 5A and FIG. 5B) and isolated according to the methods of presentinvention (FIG. 5C and FIG. 5D) at the magnifications shown in thepanels.

FIG. 6A-FIG. 6D show electron micrographs of microvesicles derived frommedium conditioned using porcine bone marrow-derived mesenchymal stemcells isolated by the ultracentrifuge method described in Example 1(FIG. 6A and FIG. 6B) and isolated according to the methods of thepresent invention (FIG. 6C and FIG. 6D) at the manifications shown inthe panels.

FIG. 7A-FIG. 7D show electron micrographs of microvesicles derived frommedium conditioned using murine bone marrow-derived mesenchymal stemcells isolated by the ultracentrifuge method described in Example 1(FIG. 7A and FIG. 7B) and isolated according to the methods of thepresent invention (FIG. 7C and FIG. 7D) at the magnifications shown inthe panels.

FIG. 8A-FIG. 8C show electron micrographs of microvesicles isolated fromhuman plasma according to the methods of the present invention. FIG. 8A,FIG. 8B, and FIG. 8C show microvesicles under the increasingmagnification, as shown by the scale bars in the panels.

FIG. 9A-FIG. 9C show electron micrographs of microvesicles isolated fromporcine plasma according to the methods of the present invention. FIG.9A, FIG. 9B, and FIG. 9C show the microvesicles under increasingmagnification, as shown by the scale bars in the panels.

FIG. 10A-FIG. 10 (C show electron micrographs of microvesicles isolatedfrom human urine according to the methods of the present invention. FIG.10A, FIG. 10B, and FIG. 10C show the microvesicles under increasingmagnification, as shown by the scale bars in the panels.

FIG. 11 shows a western blot, reporting the expression of HSP70, CD63,STAT 3 and phosphorylated STAT3 in lysates of human bone marrow-derivedmesenchymal stem cells, microvesicles isolated from medium conditionedusing human bone marrow-derived stem cells, prepared byultracentrifugation (hMSC MV Ultracentrifuge), or the methods of thepresent invention, as described in Example 3 (hMSC PEG Precipitation).Microvesicles derived from human plasma and human urine, prepared by themethods of the present invention, as described in Example 3 were alsoanalyzed. (Human plasma PEG Precipitation) and (human urine PEGPrecipitation) respectively.

FIG. 12A-FIG. 12C show the effect of microvesicles isolated from mediumconditioned using human bone marrow-derived mesenchymal stem cells onthe proliferation of normal human dermal fibroblasts (FIG. 12A), dermalfibroblasts obtained from a diabetic foot ulcer (FIG. 12B), and dermalfibroblasts obtained from a pressure foot ulcer (FIG. 12C). The effectof microvesicles isolated by ultracentrifugation (MV U/C) andmicrovesicles isolated by the methods of the present invention (MV PEG)were compared. Fibroblasts treated with PBS or microvesicle depletedculture medium were included as a control. Proliferation was determinedusing an MTT assay.

FIG. 13 shows the effect of microvesicles isolated from mediumconditioned using human bone marrow-derived mesenchymal stem cells onthe migration of human dermal fibroblasts, as determined by the abilityof the fibroblasts to migrate into a region that had been scratched off.The panel labeled “pretreatment” shows a representative area of a cellculture plate where the cells were removed, prior to the addition of thetest treatments. The effect of fibroblast migration was tested usingmicrovesicles isolated according to the methods of the present invention(PEG precipitation) and microvesicles isolated by ultracentrifugation(Ultracentrifuge) at the concentrations shown. Fibroblasts treated withPBS or microvesicle depleted culture medium were included as a control.

FIG. 14 shows the effect of microvesicles isolated from mediumconditioned using human bone marrow-derived mesenchymal stem cells onthe migration of human dermal fibroblasts obtained from a diabetic footulcer, as determined by the ability of the fibroblasts to migrate into aregion that had been scratched off. The panel labeled “pretreatment”shows a representative area of a cell culture plate where the cells wereremoved, prior to the addition of the test treatments. The effect offibroblast migration was tested using microvesicles isolated accordingto the methods of the present invention (PEG precipitation) andmicrovesicles isolated by ultracentrifugation (Ultracentrifuge) at theconcentrations shown. Fibroblasts treated with PBS or microvesicledepleted culture medium were included as a control.

FIG. 15 shows the uptake of the microvesicles of the present inventioninto human dermal fibroblasts. Cell nuclei, resolved using Hoechst 33342dye are shown in the panels labeled “Hoechst33342”. Cells, resolvedusing vybrant dye are shown in the panel labeled “Vybrant-Dio”.Microvesicles, resolved using PKH dye are shown in the panel labeled“PKH labeled MV”. A panel where images obtained from all three dyes areoverlaid is seen in the panel labeled “Composite”.

FIG. 16 shows the uptake of the microvesicles of the present inventioninto human dermal fibroblasts. Cell nuclei, resolved using Hoechst 33342dye are shown in the panels labeled “Hoechst33342”. Cells, resolvedusing vybrant dye are shown in the panel labeled “Vybrant-Dio”.Microvesicles, resolved using PKH dye are shown in the panel labeled“PKH labeled MV”. A panel where images obtained from all three dyes areoverlaid is seen in the panel labeled “Composite”.

FIG. 17 shows a western blot of lysates of human dermal fibroblaststreated with: microvesicles isolated according to the methods of thepresent invention from plasma obtained from a patient suffering fromrheumatoid arthritis (Human Plasma MV PEG Precipitation); microvesiclesisolated according to the methods of the present invention from mediumconditioned with bone marrow-derived mesenchymal stem cells (Human hMSCMXV PEG Precipitation); microvesicles isolated via ultracentrifugationfrom medium conditioned with bone marrow-derived mesenchymal stem cells(Human hMSC MV ultracentrifugation); PBS control; and a depleted mediumcontrol (hMSC conditioned medium depleted of MV).

FIG. 18 shows the presence of the region containing exon 15 of BRLAFcontaining the T1799A mutation, in: SK-MEL28 cells, from RNA amplifiedusing primer 1 (lane 3); SK-MEL28 cells, from RNA amplified using primer2 (lane 4); microvesicles isolated according to the methods of thepresent invention from medium conditioned with SK-MEL28 cells, from RNAamplified using primer 1 (lane 5); microvesicles isolated according tothe methods of the present invention from medium conditioned withSK-MEL28 cells, from RNA amplified using primer 2 (lane 6); SK-MEL28cells, from DNA amplified using primer 1 (lane 7); SK-MEL28 cells, fromDNA amplified using primer 2 (lane 8); microvesicles isolated accordingto the methods of the present invention from medium conditioned withSK-MEL28 cells, from DNA amplified using primer 1 (lane 9); andmicrovesicles isolated according to the methods of the present inventionfrom medium conditioned with SK-MEL28 cells, from DNA amplified usingprimer 2 (lane 10).

FIG. 19 shows the presence of V600E BRAF in a lysate of SK-MEL128 cellsand a lysate of microvesicles isolated according to the methods of thepresent invention from medium conditioned with SK-MEL28 cells.

FIG. 20 shows the uptake of the microvesicles isolated according to themethods of the present invention from culture medium conditioned usingbone marrow-derived stem cells obtained from a green fluorescent protein(GFP) expressing mouse into human dermal fibroblasts. Cell nuclei,resolved using Hoechst 33342 dye are shown in the panels labeled“Hoechst33342”. Cells, resolved using vybrant dye are shown in the panellabeled “Vybrant-Dio”. GFP-labeled microvesicles are shown in the panellabeled “GFP”. A panel where images obtained from all three dyes areoverlaid is seen in the panel labeled “Composite”.

FIG. 21 shows the uptake of the microvesicles isolated according to themethods of the present invention from culture medium conditioned usingbone marrow-derived stem cells obtained from a GFP expressing mouse intohuman dermal fibroblasts. Cell nuclei, resolved using Hoechst 33342 dyeare shown in the panels labeled “Hoechst33342”. Cells, resolved usingvybrant dye are shown in the panel labeled “Vybrant-Dio”. GFP-labeledmicrovesicles are shown in the panel labeled “GFP”. A panel where imagesobtained from all three dyes are overlaid is seen in the panel labeled“Composite”.

FIG. 22A-FIG. 22D show histological sections of full-thickness woundsfrom: (FIG. 22A) untreated animals; (FIG. 22B) microvesicles isolatedfrom medium conditioned using autologous bone marrow-derived mesenchymalstem cells according to the methods of the present invention; (FIG. 22C)saline: and (FIG. 22D) microvesicles isolated from autologous bonemarrow-derived mesenchymal stem cells by ultracentrifugation, 5 dayspost wound.

FIG. 23A-FIG. 23D show pictures of second degree burns on animalstreated with: (FIG. 23A) microvesicles isolated from medium conditionedusing autologous bone marrow-derived mesenchymal stem cells byultracentrifugation; (FIG. 23B) microvesicles isolated from mediumconditioned using autologous bone marrow-derived mesenchymal stem cellsaccording to the methods of the present invention; and (FIG. 23C)untreated animals, 7 days post wound. (FIG. 23D) shows a full thicknesswound in an animal treated with microvesicles isolated from mediumconditioned using autologous bone marrow-derived mesenchymal stem cellsby ultracentrifugation 7 days post wound. Arrows indicate abscessformation in a full thickness wound treated with microvesicles isolatedby ultracentrifugation at Day 7 (40×). This was not observed in fullthickness wounds treated with microvesicles prepared according to themethods of the present invention.

FIG. 24 shows a histological slide of a second degree wound, 28 dayspost wound, from an animal treated with microvesicles isolated frommedium conditioned using autologous bone marrow-derived mesenchymal stemcells according to the methods of the present invention.

FIG. 25 shows a histological slide of a second-degree wound, 28 dayspost wound, from an animal treated with saline.

FIG. 26 shows a histological slide of a full-thickness wound, 28 dayspost wound, from an animal treated with microvesicles isolated frommedium conditioned using autologous bone marrow-derived mesenchymal stemcells according to the methods of the present invention.

FIG. 27A-FIG. 27C show a histological slide of a full-thickness wound,28 days post wound, from an animal treated with microvesicles isolatedfrom medium conditioned using autologous bone marrow-derived mesenchymalstem cells according to the methods of the present invention. FIG. 27Ashows new nerve growth (arrows) and angiogenesis (circles). FIG. 27Bshows new nerve growth (arrows). FIG. 27C shows new blood vessel growth(arrows).

FIG. 28 shows a histological slide of a full-thickness wound, 7 dayspost wound in an animal treated with microvesicles derived from mediumconditioned using autologous bone marrow-derived mesenchymal stem cells.

FIG. 29A-FIG. 29B shows the presence or absence of chimerism inirradiated animals administration of GFP-labeled bone marrow.

FIG. 30A-FIG. 30B show the effects of MSC treatment on hair growthfollowing gamma irradiation.

FIG. 30C shows the absence of chimerism in irradiated animals followingadministration of GFP-labeled bone marrow.

FIG. 31 shows the effect of bone marrow-derived microvesicles obtainedusing the method of the present invention on blood vessel formation,using an in vitro assay of angiogenesis. The upper three panels arerepresentative images taken using an epifluorescent microscope ofcultures of HUVEC cells treated with bone marrow-derived microvesiclesobtained using the method of the present invention (“Bone MarrowAspriate MV”). The lower three panels are representative images takenusing an epifluorescent microscope of cultures of HUVEC cells treatedwith vehicle control (“Vehicle Control”).

FIG. 32A-FIG. 32B show the effect of bone marrow-derived microvesiclesobtained using the method of the present invention on cell growth orproliferation, using an in vitro assay of cell growth. FIG. 32A showsrepresentative images taken using an epifluorescent microscope ofcultures of normal adult fibroblasts treated with bone marrow-derivedmicrovesicles obtained method of the present invention (“Bone MarrowMV”) or PBS (“PBS”), three days post treatment. FIG. 32B shows theaverage cell number in cultures of normal adult fibroblasts treated withbone marrow-derived microvesicles obtained using the method of thepresent invention (“Bone Marrow MV”) or PBS (“PBS”), three days posttreatment.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionsthat describe or illustrate certain features, embodiments orapplications of the present invention.

The Methods to Isolate the Microvesicles of the Present Invention

In one embodiment, microvesicles are isolated from a biological fluidcontaining microvesicles in a method comprising the steps of:

-   -   a) obtaining a biological fluid containing microvesicles,    -   b) clarifying the biological fluid to remove cellular debris,    -   c) precipitating the microvesicles by adding a precipitating        agent to the clarified biological fluid,    -   d) collecting the precipitated microvesicles and washing the        material to remove the precipitating agent, and    -   e) suspending the washed microvesicles in a solution for storage        or subsequent use.

In one embodiment, the biological fluid is clarified by centrifugation.In an alternate embodiment, the biological fluid is clarified byfiltration.

In one embodiment, the precipitated microvesicles are collected bycentrifugation. In an alternate embodiment, the precipitatedmicrovesicles are collected by filtration.

In one embodiment, microvesicles are isolated from a biological fluidcontaining microvesicles in a method comprising the steps of:

-   -   a) obtaining a biological fluid containing microvesicles,    -   b) clarifying the biological fluid to remove cellular debris,    -   c) precipitating the microvesicles by adding a precipitating        agent to the clarified biological fluid,    -   d) collecting the precipitated microvesicles and washing the        material to remove the precipitating agent,    -   e) suspending the washed microvesicles in a solution, and    -   f) processing the microvesicles to analyze the nucleic acid,        carbohydrate, lipid, small molecules and/or protein content.

In one embodiment, the biological fluid is clarified by centrifugation.In an alternate embodiment, the biological fluid is clarified byfiltration.

In one embodiment, the precipitated microvesicles are collected bycentrifugation. In an alternate embodiment, the precipitatedmicrovesicles are collected by filtration.

In one embodiment, the present invention provides reagents and kits toisolate microvesicles from biological fluids according to the methods ofthe present invention.

The biological fluid may be peripheral blood, sera, plasma, ascites,urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovialfluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen (including prostatic fluid),Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecalmatter, hair, tears, cyst fluid, pleural and peritoneal fluid,pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid,menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stoolwater, pancreatic juice, lavage fluids from sinus cavities,bronchopulmonary aspirates or other lavage fluids.

The biological fluid may also be derived from the blastocyl cavity,umbilical cord blood, or maternal circulation, which may be of fetal ormaternal origin. The biological fluid may also be derived from a tissuesample or biopsy.

The biological fluid may be derived from plant cells of cultures ofplant cells. The biological fluid may be derived from yeast cells orcultures of yeast cells.

In one embodiment, the biological fluid is cell culture medium. In oneembodiment, the cell culture medium is conditioned using tissues and/orcells prior to the isolation of microvesicles according to the methodsof the present invention.

The term “conditioned” or “conditioned medium” refers to medium, whereina population of cells or tissue, or combination thereof is grown, andthe population of cells or tissue, or combination thereof contributesfactors to the medium. In one such use, the population of cells ortissue, or combination thereof is removed from the medium, while thefactors the cells produce remain. In one embodiment, the factorsproduced are microvesicles. Medium may be conditioned via any suitablemethod selected by one of ordinary skill in the art. For example, mediummay be cultured according to the methods described in EP1780267A2.

In one embodiment, microvesicles are isolated from cells or tissue thathave been pre-treated prior to the isolation of the microvesicles.Pretreatment may include, for example, culture in a specific medium, amedium that contains at least one additive, growth factor, medium devoidof serum, or a combination thereof. Alternatively, pretreatment maycomprise contacting cells or tissues with additives (e.g. interleukin,VEGF, inducers of transcription factors, transcription factors,hormones, neurotransmitters, pharmaceutical compounds, microRNA),transforming agents (e.g. liposome, viruses, transfected agents, etc.).Alternatively, pretreatment may comprise exposing cells or tissue toaltered physical conditions (e.g. hypoxia, cold shock, heat shock).

In one embodiment, microvesicles are isolated from medium conditionedusing cells or tissue that have been pre-treated prior to the isolationof the microvesicles. Pretreatment may include, for example, culture ina specific medium, a medium that contains at least one additive, growthfactor, medium devoid of serum, or a combination thereof. Alternatively,pretreatment may comprise contacting cells or tissues with additives(e.g. interleukin, VEGF, inducers of transcription factors,transcription factors, hormones, neurotransmitters, pharmaceuticalcompounds, microRNA), transforming agents (e.g. liposome, viruses,transfected agents, etc.). Alternatively, pretreatment may compriseexposing cells or tissue to altered physical conditions (e.g. hypoxia,cold shock, heat shock).

In one embodiment, the biological fluid is an extract from a plant. Inan alternate embodiment, the biological fluid is a cell culture mediumfrom a culture of plant cells. In an alternate embodiment, thebiological fluid is yeast extract. In an alternate embodiment, thebiological fluid is a cell culture medium from a culture of yeast cells.

While the methods of the present invention may be carried out at anytemperature, one of ordinary skill in the art can readily appreciatethat certain biological fluids may degrade, and such degradation isreduced if the sample is maintained at a temperature below thetemperature at which the biological fluid degrades. In one embodiment,the method of the present invention is carried out at 4° C. In analternate embodiment, at least one step of the method of the presentinvention is carried out at 4° C.

In certain embodiments, the biological fluid may be diluted prior tobeing subjected to the methods of the present invention. Dilution may berequired for viscous biological fluids, to reduce the viscosity of thesample, if the viscosity of the sample is too great to obtain anacceptable yield of microvesicles. The dilution may be a 1:2 dilution.Alternatively, the dilution may be a 1:3 dilution. Alternatively, thedilution may be a 1:4 dilution. Alternatively, the dilution may be a 1:5dilution. Alternatively, the dilution may be a 1:6 dilution.Alternatively, the dilution may be a 1:7 dilution. Alternatively, thedilution may be a 1:8 dilution. Alternatively, the dilution may be a 1:9dilution. Alternatively, the dilution may be a 1:10 dilution.Alternatively, the dilution may be a 1:20 dilution. Alternatively, thedilution may be a 1:30 dilution. Alternatively, the dilution may be a1:40 dilution. Alternatively, the dilution may be a 1:50 dilution.Alternatively, the dilution may be a 1:60 dilution. Alternatively, thedilution may be a 1:70 dilution. Alternatively, the dilution may be a1:80 dilution. Alternatively, the dilution may be a 1:90 dilution.Alternatively, the dilution may be a 1:100 dilution.

The biological fluid may be diluted with any diluent, provided thediluent does not affect the functional and/or structural integrity ofthe microvesicles. One of ordinary skill in the art may readily select asuitable diluent. Diluents may be, for example, phosphate bufferedsaline, cell culture medium, and the like.

In one embodiment, the biological fluid is clarified by the applicationof a centrifugal force to remove cellular debris. The centrifugal forceapplied to the biological fluid is sufficient to remove any cells, lysedcells, tissue debris from the biological fluid, but the centrifugalforce applied is insufficient in magnitude, duration, or both, to removethe microvesicles. The biological fluid may require dilution tofacilitate the clarification.

The duration and magnitude of the centrifugal force used to clarify thebiological fluid may vary according to a number of factors readilyappreciated by one of ordinary skill in the art, including, for example,the biological fluid, the pH of the biological fluid, the desired purityof the isolated microvesicles, the desired size of the isolatedmicrovesicles, the desired molecular weight of the microvesicles, andthe like. In one embodiment, a centrifugal force of 2000×g is applied tothe biological fluid for 30 minutes.

The clarified biological fluid is contacted with a precipitation agentto precipitate the microvesicles. In one embodiment, the precipitationagent may be any agent that surrounds the microvesicles and displacesthe water of solvation. Such precipitation agents may be selected fromthe group consisting of polyethylene glycol, dextran, andpolysaccharides.

In an alternate embodiment, the precipitation agent may causeaggregation of the microvesicles.

In an alternate embodiment, the precipitation agent is selected from thegroup consisting of calcium ions, magnesium ions, sodium ions, ammoniumions, iron ions, organic solvents such as ammonium sulphate, andflocculating agents, such as alginate.

The clarified biological fluid is contacted with the precipitation agentfor a period of time sufficient to precipitate the microvesicles. Theperiod of time sufficient to precipitate the microvesicles may varyaccording to a number of factors readily appreciated by one of ordinaryskill in the art, including, for example, the biological fluid, the pHof the biological fluid, the desired purity of the isolatedmicrovesicles, the desired size of the isolated microvesicles, thedesired molecular weight of the microvesicles, and the like. In oneembodiment, the period of time sufficient to precipitate themicrovesicles is 6 hours.

In one embodiment, the clarified biological fluid is contacted with theprecipitation agent for a period of time sufficient to precipitate themicrovesicles at 4° C.

The concentration of the precipitation agent used to precipitate themicrovesicles from a biological fluid may vary according to a number offactors readily appreciated by one of ordinary skill in the art,including, for example, the biological fluid, the pH of the biologicalfluid, the desired purity of the isolated microvesicles, the desiredsize of the isolated microvesicles, the desired molecular weight of themicrovesicles, and the like.

In one embodiment, the precipitation agent is polyethylene glycol. Themolecular weight of polyethylene glycol used in the methods of thepresent invention may be from about 200 Da to about 10,000 Da. In oneembodiment, the molecular weight of polyethylene glycol used in themethods of the present invention may be greater than 10,000 Da. Thechoice of molecular weight may be influenced by a variety of factorsincluding, for example, the viscosity of the biological fluid, thedesired purity of the microvesicles, the desired size of themicrovesicles, the biological fluid used, and the like.

In one embodiment, the molecular weight of polyethylene glycol used inthe methods of the present invention may be from about 200 Da to about8,000 Da, or is approximately any of 200, 300, 400, 600, 1000, 1500,4000, 6000, or 8000.

In one embodiment, the molecular weight of polyethylene glycol used inthe methods of the present invention is about 6000 Da.

In one embodiment, the average molecular weight of polyethylene glycolused in the methods of the present invention is about 8000 Da.

The concentration of polyethylene glycol used in the methods of thepresent invention may be from about 0.5% w/v to about 100% w/v. Theconcentration of polyethylene glycol used in the methods of the presentinvention may be influenced by a variety of factors including, forexample, the viscosity of the biological fluid, the desired purity ofthe microvesicles, the desired size of the microvesicles, the biologicalfluid used, and the like.

In certain embodiments, the polyethylene glycol is used in theconcentration of the present invention at a concentration between about5% and 25% w/v. In certain embodiments, the concentration is about 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or a range between anytwo of these values.

In one embodiment, the concentration of polyethylene glycol used in themethods of the present invention is about 8.5% w/v.

In one embodiment, the concentration of polyethylene glycol used in themethods of the present invention is about 6% w/v.

In one embodiment, polyethylene glycol having an average molecularweight of 6000 Da is used, at a concentration of 8.5% w/v. In oneembodiment, the polyethylene glycol is diluted in 0.4M sodium chloride.

In one embodiment, the concentration of the polyethylene glycol used inthe methods of the present invention is inversely proportional to theaverage molecular weight of the polyethylene glycol. For example, in oneembodiment, polyethylene glycol having an average molecular weight of4000 Da is used, at a concentration of 20% w/v. In an alternateembodiment, polyethylene glycol having an average molecular weight of8000 Da is used, at a concentration of 10% w/v. In an alternateembodiment, polyethylene glycol having an average molecular weight of20000 Da is used, at a concentration of 4% w/v.

In one embodiment, the precipitated microvesicles are collected by theapplication of centrifugal force. The centrifugal force is sufficientand applied for a duration sufficient to cause the microvesicles to forma pellet, but insufficient to damage the microvesicles.

The duration and magnitude of the centrifugal force used to precipitatethe microvesicles from a biological fluid may vary according to a numberof factors readily appreciated by one of ordinary skill in the art,including, for example, the biological fluid, the pH of the biologicalfluid, the desired purity of the isolated microvesicles, the desiredsize of the isolated microvesicles, the desired molecular weight of themicrovesicles, and the like. In one embodiment, the precipitatedmicrovesicles are collected by the application of a centrifugal force of10000×g for 60 minutes.

The precipitated microvesicles may be washed with any liquid, providedthe liquid does not affect the functional and/or structural integrity ofthe microvesicles. One of ordinary skill in the art may readily select asuitable liquid. Liquids may be, for example, phosphate buffered saline,cell culture medium, and the like.

In one embodiment, the washing step removes the precipitating agent. Inone embodiment, the microvesicles are washed via centrifugal filtration,using a filtration device with a 100 kDa molecular weight cut off.

The isolated microvesicles may be suspended with any liquid, providedthe liquid does not affect the functional and/or structural integrity ofthe microvesicles. One of ordinary skill in the art may readily select asuitable liquid. Liquids may be, for example, phosphate buffered saline,cell culture medium, and the like.

In one embodiment, the isolated microvesicles may be further processed.The further processing may be the isolation of a microvesicle of aspecific size. Alternatively, the further processing may be theisolation of microvesicles of a particular size range. Alternatively,the further processing may be the isolation of a microvesicle of aparticular molecular weight. Alternatively, the further processing maybe the isolation of microvesicles of a particular molecular weightrange. Alternatively, the further processing may be the isolation of amicrovesicle exhibiting or containing a specific molecule.

In one embodiment, the microvesicles of the present invention arefurther processed to isolate a preparation of microvesicles having asize of about 2 nm to about 1000 nm as determined by electronmicroscopy. In an alternate embodiment, the microvesicles of the presentinvention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 500 nm as determinedby electron microscopy. In an alternate embodiment, the microvesicles ofthe present invention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 400 nm as determinedby electron microscopy. In an alternate embodiment, the microvesicles ofthe present invention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 300 nm as determinedby electron microscopy. In an alternate embodiment, the microvesicles ofthe present invention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 200 nm as determinedby electron microscopy. In an alternate embodiment, the microvesicles ofthe present invention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 100 nm as determinedby electron microscopy. In an alternate embodiment, the microvesicles ofthe present invention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 50 nm as determinedby electron microscopy. In an alternate embodiment, the microvesicles ofthe present invention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 20 nm as determinedby electron microscopy. In an alternate embodiment, the microvesicles ofthe present invention are further processed to isolate a preparation ofmicrovesicles having a size of about 2 nm to about 10 nm as determinedby electron microscopy.

In one embodiment, the subsequent purification is performed using amethod selecting from the group consisting of immunoaffinity, HPLC,tangential flow filtration, phase separation/partitioning, andmicrofluidics.

In one embodiment, the isolated microvesicles are further processed toanalyze the molecules exhibited on, or contained within themicrovesicles. The molecules analyzed are selected from the groupconsisting of nucleic acid, carbohydrate, lipid, small molecules, ions,metabolites, protein, and combinations thereof.

Biological Fluid Comprising Cell Culture Medium Conditioned UsingCultured Cells:

In one embodiment, microvesicles are obtained from medium conditionedusing cultured cells. Any cultured cell, or population of cells may beused in the methods of the present invention. The cells may be stemcells, primary cells, cell lines, tissue or organ explants, or anycombination thereof. The cells may be allogeneic, autologous, orxenogeneic in origin.

In one embodiment, the cells are cells derived from bone-marrowaspirate. In one embodiment, the cells derived from bone marrow aspirateare bone marrow-derived mesenchymal stem cells. In one embodiment, thecells derived from bone marrow aspirate are mononuclear cells. In oneembodiment, the cells derived from bone marrow aspirate are a mixture ofmononuclear cells and bone marrow-derived mesenchymal stem cells.

In one embodiment, bone marrow-derived mesenchymal stem cells areisolated from bone marrow aspirate by culturing bone marrow aspirate inplastic tissue culture flasks for a period of time of up to about 4days, followed by a wash to remove the non-adherent cells.

In one embodiment, mononuclear cells are isolated from bone marrowaspirate by low-density centrifugation using a ficoll gradient, andcollecting the mononuclear cells at the interface.

In one embodiment, prior to isolation of microvesicles according to themethods of the present invention, the cells are cultured, grown ormaintained at an appropriate temperature and gas mixture (typically, 37°C., 5% CO₂ for mammalian cells) in a cell incubator. Culture conditionsvary widely for each cell type, and are readily determined by one ofordinary skill in the art.

In one embodiment, one, or more than one culture condition is varied. Inone embodiment, this variation results in a different phenotype.

In one embodiment, where the cells require serum in their culturemedium, to begin the microvesicle isolation procedure, the cell culturemedium is supplemented with microvesicle-free serum and then added tothe cells to be conditioned. The microvesicles are collected from theconditioned cell culture medium. Serum may be depleted by any suitablemethod, such as, for example, ultracentrifugation, filtration,precipitation, and the like. The choice of medium, serum concentration,and culture conditions are influenced by a variety of factors readilyappreciated by one of ordinary skill in the art, including, for example,the cell type being cultured, the desired purity of the microvesicles,the desired phenotype of the cultured cell, and the like. In oneembodiment, the cell culture medium that is conditioned for themicrovesicle isolation procedure is the same type of cell culture mediumthat the cells were grown in, prior to the microvesicle isolationprocedure.

In one embodiment, to begin the microvesicle isolation procedure, thecell culture medium is removed, and serum-free medium is added to thecells to be conditioned. The microvesicles are then collected from theconditioned serum free medium. The choice of medium, and cultureconditions are influenced by a variety of factors readily appreciated byone of ordinary skill in the art, including, for example, the cell typebeing cultured, the desired purity of the microvesicles, the desiredphenotype of the cultured cell, and the like. In one embodiment, theserum-free medium is supplemented with at least one additional factorthat promotes or enhances the survival of the cells in the serum freemedium. Such factor may, for example, provide trophic support to thecells, inhibit, or prevent apoptosis of the cells.

The cells are cultured in the culture medium for a period of timesufficient to allow the cells to secrete microvesicles into the culturemedium. The period of time sufficient to allow the cells to secretemicrovesicles into the culture medium is influenced by a variety offactors readily appreciated by one of ordinary skill in the art,including, for example, the cell type being cultured, the desired purityof the microvesicles, the desired phenotype of the cultured cell,desired yield of microvesicles, and the like.

The microvesicles are then removed from the culture medium by themethods of the present invention.

In one embodiment, prior to the microvesicle isolation procedure, thecells are treated with at least one agent selected from the groupconsisting of an anti-inflammatory compound, an anti-apoptotic compound,an inhibitor of fibrosis, a compound that is capable of enhancingangiogenesis, an immunosuppressive compound, a compound that promotessurvival of the cells, a chemotherapeutic, a compound capable ofenhancing cellular migration, a neurogenic compound, and a growthfactor.

In one embodiment, while the cells are being cultured in the medium fromwhich the microvesicles are collected, the cells are treated with atleast one agent selected from the group consisting of ananti-inflammatory compound, an anti-apoptotic compound, an inhibitor offibrosis, a compound that is capable of enhancing angiogenesis, animmunosuppressive compound, a compound that promotes survival of thecells, and a growth factor.

In one embodiment, the anti-inflammatory compound may be selected fromthe compounds disclosed in U.S. Pat. No. 6,509,369.

In one embodiment, the anti-apoptotic compound may be selected from thecompounds disclosed in U.S. Pat. No. 6,793,945.

In one embodiment, the inhibitor of fibrosis may be selected from thecompounds disclosed in U.S. Pat. No. 6,331,298.

In one embodiment, the compound that is capable of enhancingangiogenesis may be selected from the compounds disclosed in U.S. PatentApplication 2004/0220393 or U.S. Patent Application 2004/0209901.

In one embodiment, the immunosuppressive compound may be selected fromthe compounds disclosed in U.S. Patent Application 2004/0171623.

In one embodiment, the compound that promotes survival of the cells maybe selected from the compounds disclosed in U.S. Patent Application2010/0104542.

In one embodiment, the growth factor may be at least one moleculeselected from the group consisting of members of the TGF-β family,including TGF-β1, 2, and 3, bone morphogenic proteins (BMP-2, -3,-4, -5,-6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2,platelet-derived growth factor-AA, -AB, and -BB, platelet rich plasma,insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5,-6, -8, -10, -15), vascular endothelial cell-derived growth factor(VEGF), pleiotrophin, endothelin, among others. Other pharmaceuticalcompounds can include, for example, nicotinamide, hypoxia induciblefactor 1-alpha, glucagon like peptide-1 (GLP-1), GLP-1 and GLP-2mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid,parathyroid hormone, tenascin-C, tropoelastin, thrombin-derivedpeptides, cathelicidins, defensins, laminin, biological peptidescontaining cell- and heparin-binding domains of adhesive extracellularmatrix proteins such as fibronectin and vitronectin, and MAPKinhibitors, such as, for example, compounds disclosed in U.S. PatentApplication 2004/0209901 and U.S. Patent Application 2004/0132729.

In one embodiment, microvesicles are isolated from a biological fluidcomprising cell culture medium conditioned using a culture of bonemarrow-derived mesenchymal stem cells comprising the steps of:

-   -   a) obtaining a population of bone marrow-derived mesenchymal        stem cells and seeding flasks at a 1:4 dilution of cells,    -   b) culturing the cells in medium until the cells are 80 to 90%        confluent,    -   c) removing and clarifying the medium to remove cellular debris,    -   d) precipitating the microvesicles by adding a precipitating        agent to the clarified culture medium,    -   e) collecting the precipitated microvesicles and washing the        material to remove the precipitating agent, and    -   f) suspending the washed microvesicles in a solution for storage        or subsequent use.

In one embodiment, microvesicles are isolated from a biological fluidcomprising cell culture medium conditioned using a culture of bonemarrow-derived mononuclear cells comprising the steps of:

-   -   a) obtaining a population of bone marrow-derived mononuclear        cells and seeding flasks at a 1:4 dilution of cells,    -   b) culturing the cells in medium until the cells are 80 to 90%        confluent,    -   c) removing and clarifying the medium to remove cellular debris,    -   d) precipitating the microvesicles by adding a precipitating        agent to the clarified culture medium,    -   e) collecting the precipitated microvesicles and washing the        material to remove the precipitating agent, and    -   f) suspending the washed microvesicles in a solution for storage        or subsequent use.

In one embodiment, the bone marrow-derived mesenchymal stem cells arecultured in medium comprising α-MEM supplemented with 20% fetal bovineserum and 1% penicillin/streptomycin/glutamine at 37° C. in 95%humidified air and 5% CO₂.

In one embodiment, the bone marrow-derived mononuclear cells arecultured in medium comprising α-MEM supplemented with 20% fetal bovineserum and 1% penicillin/streptomycin/glutamine at 37° C. in 95%humidified air and 5% CO₂.

In one embodiment, the medium is clarified by centrifugation.

In one embodiment, the precipitating agent is polyethylene glycol havingan average molecular weight of 6000. In one embodiment, the polyethyleneglycol is used at a concentration of about 8.5 w/v %. In one embodiment,the polyethylene glycol is diluted in a sodium chloride solution havinga final concentration of 0.4 M.

In one embodiment, the precipitated microvesicles are collected bycentrifugation.

In one embodiment, the isolated microvesicles are washed via centrifugalfiltration, using a membrane with a 100 kDa molecular weight cut-off,using phosphate buffered saline.

Biological fluid comprising plasma: In one embodiment, microvesicles areobtained from plasma. The plasma may be obtained from a healthyindividual, or, alternatively, from an individual with a particulardisease phenotype.

In one embodiment, microvesicles are isolated from a biological fluidcomprising plasma comprising the steps of:

-   -   a) obtaining plasma and diluting the plasma with cell culture        medium,    -   b) precipitating the microvesicles by adding a precipitating        agent to the diluted plasma,    -   c) collecting the precipitated microvesicles and washing the        material to remove the precipitating agent, and    -   d) suspending the washed microvesicles in a solution for storage        or subsequent use.

In one embodiment, the plasma is diluted 1:10 with culture medium.

In one embodiment, the culture medium is α-MEM.

In one embodiment, the precipitating agent is polyethylene glycol havingan average molecular weight of 6000. In one embodiment, the polyethyleneglycol is used at a concentration of about 8.5 w/v %. In one embodiment,the polyethylene glycol is diluted in a sodium chloride solution havinga final concentration of 0.4 M.

In one embodiment, the precipitated microvesicles are collected bycentrifugation.

In one embodiment, the isolated microvesicles are washed via centrifugalfiltration, using a membrane with a 100 kDa molecular weight cut-off,using phosphate buffered saline.

Biological fluid comprising bone marrow aspirate: In one embodiment,microvesicles are obtained from bone marrow aspirate. In one embodiment,microvesicles are obtained from the cellular fraction of the bone marrowaspirate. In one embodiment, microvesicles are obtained from theacellular fraction of the bone marrow aspirate.

In one embodiment, microvesicles are obtained from cells cultured frombone marrow aspirate. In one embodiment, the cells cultured from bonemarrow aspirate are used to condition cell culture medium, from whichthe microvesicles are isolated.

In one embodiment, microvesicles are isolated from a biological fluidcomprising bone marrow aspirate comprising the steps of:

-   -   a) obtaining bone marrow aspirate and separating the bone marrow        aspirate into an acellular portion and a cellular portion,    -   b) diluting the acellular portion,    -   c) clarifying the diluted acellular portion to remove cellular        debris,    -   d) precipitating the microvesicles in the acellular portion by        adding a precipitating agent to the diluted acellular portion,    -   e) collecting the precipitated microvesicles and washing the        material to remove the precipitating agent, and    -   f) suspending the washed microvesicles in a solution for storage        or subsequent use.

In one embodiment, the acellular portion is diluted 1:10 with culturemedium.

In one embodiment, the culture medium is α-MEM.

In one embodiment, the diluted acellular portion is clarified bycentrifugation.

In one embodiment, the precipitating agent is polyethylene glycol havingan average molecular weight of 6000. In one embodiment, the polyethyleneglycol is used at a concentration of about 8.5 w/v %. In one embodiment,the polyethylene glycol is diluted in a sodium chloride solution havinga final concentration of 0.4 M.

In one embodiment, the precipitated microvesicles are collected bycentrifugation.

In one embodiment, the isolated microvesicles are washed via centrifugalfiltration, using a membrane with a 100 kDa molecular weight cut-off,using phosphate buffered saline.

In one embodiment the cellular portion is further processed to isolateand collect cells. In one embodiment, the cellular portion is furtherprocessed to isolate and collect bone marrow-derived mesenchymal stemcells. In one embodiment, the cellular portion is further processed toisolate and collect bone marrow-derived mononuclear cells. In oneembodiment, the cellular portion is used to condition medium, from whichmicrovesicles may later be derived.

In one embodiment, microvesicles are isolated from the cellular portion.The cellular portion may be incubated for a period of time prior to theisolation of the microvesicles.

Alternatively, the microvesicles may be isolated from the cellularportion immediately after the cellular portion is collected.

In one embodiment, the cellular portion is also treated with at leastone agent selected from the group consisting of an anti-inflammatorycompound, an anti-apoptotic compound, an inhibitor of fibrosis, acompound that is capable of enhancing angiogenesis, an immunosuppressivecompound, a compound that promotes survival of the cells, achemotherapeutic, a compound capable of enhancing cellular migration, aneurogenic compound, and a growth factor.

In one embodiment, the anti-inflammatory compound may be selected fromthe compounds disclosed in U.S. Pat. No. 6,509,369.

In one embodiment, the anti-apoptotic compound may be selected from thecompounds disclosed in U.S. Pat. No. 6,793,945.

In one embodiment, the inhibitor of fibrosis may be selected from thecompounds disclosed in U.S. Pat. No. 6,331,298.

In one embodiment, the compound that is capable of enhancingangiogenesis may be selected from the compounds disclosed in U.S. PatentApplication 2004/0220393 or U.S. Patent Application 2004/0209901.

In one embodiment, the immunosuppressive compound may be selected fromthe compounds disclosed in U.S. Patent Application 2004/0171623.

In one embodiment, the compound that promotes survival of the cells maybe selected from the compounds disclosed in U.S. Patent Application2010/0104542.

In one embodiment, the growth factor may be at least one moleculeselected from the group consisting of members of the TGF-β family,including TGF-β1, 2, and 3, bone morphogenic proteins (BMP-2, -3,-4, -5,-6, -7, -11, -12, and -13), fibroblast growth factors-1 and -2,platelet-derived growth factor-AA, -AB, and -BB, platelet rich plasma,insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5,-6, -8, -10, -15), vascular endothelial cell-derived growth factor(VEGF), pleiotrophin, endothelin, among others. Other pharmaceuticalcompounds can include, for example, nicotinamide, hypoxia induciblefactor 1-alpha, glucagon like peptide-1 (GLP-1), GLP-1 and GLP-2mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid,parathyroid hormone, tenascin-C, tropoelastin, thrombin-derivedpeptides, cathelicidins, defensins, laminin, biological peptidescontaining cell- and heparin-binding domains of adhesive extracellularmatrix proteins such as fibronectin and vitronectin, and MAPKinhibitors, such as, for example, compounds disclosed in U.S. PatentApplication 2004/0209901 and U.S. Patent Application 2004/0132729.

In one embodiment, the cellular portion is cultured under hypoxicconditions.

In one embodiment, the cellular portion is heat-shocked.

Biological fluid comprising urine: In one embodiment, microvesicles areobtained from urine. The urine may be obtained from a healthyindividual, or, alternatively, from an individual with a particulardisease phenotype.

In one embodiment, microvesicles are isolated from a biological fluidcomprising urine comprising the steps of:

-   -   a) obtaining a urine sample,    -   b) clarifying the urine to remove cellular debris,    -   c) precipitating the microvesicles by adding a precipitating        agent to the clarified urine,    -   d) collecting the precipitated microvesicles and washing the        material to remove the precipitating agent, and    -   e) suspending the washed microvesicles in a solution for storage        or subsequent use.

In one embodiment, the urine is clarified by centrifugation.

In one embodiment, the precipitating agent is polyethylene glycol havingan average molecular weight of 6000. In one embodiment, the polyethyleneglycol is used at a concentration of about 8.5 w/v %. In one embodiment,the polyethylene glycol is diluted in a sodium chloride solution havinga final concentration of 0.4 M.

In one embodiment, the precipitated microvesicles are collected bycentrifugation.

In one embodiment, the isolated microvesicles are washed via centrifugalfiltration, using a membrane with a 100 kDa molecular weight cut-off,using phosphate buffered saline.

In an alternate embodiment of the present invention, the biologicalfluids are clarified by filtration. In an alternate embodiment, theprecipitated microvesicles are collected by filtration. In an alternateembodiment, the biological fluids are clarified and the precipitatedmicrovesicles are collected by filtration.

In certain embodiments, filtration of either the biological fluid,and/or the precipitated microvesicles required the application of anexternal force. The external force may be gravity, either normal gravityor centrifugal force. Alternatively, the external force may be suction.

In one embodiment, the present embodiment provides an apparatus tofacilitate the clarification of the biological fluid by filtration. Inone embodiment, the present invention provides an apparatus tofacilitate collection of the precipitated microvesicles by filtration.In one embodiment, the present invention provides an apparatus thatfacilitates the clarification of the biological fluid and the collectionof the precipitated microvesicles by filtration. In one embodiment, theapparatus also washes the microvesicles.

In one embodiment, the apparatus is the apparatus shown in FIG. 4 . Inthis embodiment, the biological fluid is added to the inner chamber. Theinner chamber has a first filter with a pore size that enables themicrovesicles to pass, while retaining any particle with a size greaterthan a microvesicle in the inner chamber. In one embodiment, the poresize of the filter of the inner chamber is 1 μm. In this embodiment,when the biological fluid passed from the inner chamber through thefilter, particles greater than 1 μm are retained in the inner chamber,and all other particles collect in the region between the bottom of theinner chamber and a second filter.

The second filter has a pore size that does not allow microvesicles topass. In one embodiment, the pore size of the second filter of the innerchamber is 0.01 km. In this embodiment, when the biological fluid passedthrough the second filter, the microvesicles are retained in the regionbetween the bottom of the inner chamber and the second filter, and allremaining particles and fluid collect in the bottom of the apparatus.

One of ordinary skill in the art can readily appreciate that theapparatus can have more than two filters, of varying pore sizes toselect for microvesicles of desired sizes, for example.

In one embodiment, a precipitating agent is added to the biologicalfluid in the inner chamber. In one embodiment, a precipitating agent isadded to the filtrate after it has passed through the first filter.

The filter membranes utilized by the apparatus of the present inventionmay be made from any suitable material, provided the filter membranedoes not react with the biological fluid, or bind with components withinthe biological fluid. For example, the filter membranes may be made froma low bind material, such as, for example, polyethersulfone, nylon6,polytetrafluoroethylene, polypropylene, zeta modified glass microfiber,cellulose nitrate, cellulose acetate, polyvinylidene fluoride,regenerated cellulose.

The Microvesicles of the Present Invention

In one embodiment, the microvesicles of the present invention have asize of about 2 nm to about 5000 nm as determined by electronmicroscopy. In an alternate embodiment, the microvesicles of the presentinvention have a size of about 2 nm to about 1000 nm as determined byelectron microscopy. In an alternate embodiment, the microvesicles ofthe present invention have a size of about 2 nm to about 500 nm asdetermined by electron microscopy. In an alternate embodiment, themicrovesicles of the present invention have a size of about 2 nm toabout 400 nm as determined by electron microscopy. In an alternateembodiment, the microvesicles of the present invention have a size ofabout 2 nm to about 300 nm as determined by electron microscopy. In analternate embodiment, the microvesicles of the present invention have asize of about 2 nm to about 200 nm as determined by electron microscopy.In an alternate embodiment, the microvesicles of the present inventionhave a size of about 2 nm to about 100 nm as determined by electronmicroscopy. In an alternate embodiment, the microvesicles of the presentinvention have a size of about 2 nm to about 50 nm as determined byelectron microscopy. In an alternate embodiment, the microvesicles ofthe present invention have a size of about 2 nm to about 20 nm asdetermined by electron microscopy. In an alternate embodiment, themicrovesicles of the present invention have a size of about 2 nm toabout 10 nm as determined by electron microscopy.

In one embodiment, the microvesicles of the present invention have amolecular weight of at least 100 kDa.

Microvesicles isolated according to the methods of the present inventionmay be used for therapies. Alternatively, microvesicles isolatedaccording to the methods of the present invention may be used fordiagnostic tests. Alternatively, the microvesicles of the presentinvention may be used to alter or engineer cells or tissues. In the casewhere the microvesicles of the present invention are used to alter orengineer cells or tissues, the microvesicles may be loaded, labeled withRNA, DNA, lipids, carbohydrates, protein, drugs, small molecules,metabolites, or combinations thereof, that will alter or engineer a cellor tissue. Alternatively, the microvesicles may be isolated from cellsor tissues that express and/or contain the RNA, DNA, lipids,carbohydrates, protein, drugs, small molecules, metabolites, orcombinations thereof.

Use of the Microvesicles of the Present Invention in Diagnostic Tests

The microvesicles of the present invention can be used in a diagnostictest that detects biomarkers that identify particular phenotypes suchas, for example, a condition or disease, or the stage or progression ofa disease. Biomarkers or markers from cell-of-origin specificmicrovesicles can be used to determine treatment regimens for diseases,conditions, disease stages, and stages of a condition, and can also beused to determine treatment efficacy. Markers from cell-of-originspecific microvesicles can also be used to identify conditions ofdiseases of unknown origin.

As used herein, the term “biomarker” refers to an indicator of abiological state. It is a characteristic that is objectively measuredand evaluated as an indicator of normal biological processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention.

One or more biomarkers of microvesicle can be assessed forcharacterizing a phenotype. The biomarker can be a metabolite, a nucleicacid, peptide, protein, lipid, antigen, carbohydrate or proteoglycan,such as DNA or RNA. The RNA can be mRNA, miRNA, snoRNA, snRNA, rRNAs,tRNAs, siRNA, hnRNA, or shRNA.

A phenotype in a subject can be characterized by obtaining a biologicalsample from the subject and analyzing one or more microvesicles from thesample. For example, characterizing a phenotype for a subject orindividual may include detecting a disease or condition (includingpre-symptomatic early stage detecting), determining the prognosis,diagnosis, or theranosis of a disease or condition, or determining thestage or progression of a disease or condition. Characterizing aphenotype can also include identifying appropriate treatments ortreatment efficacy for specific diseases, conditions, disease stages andcondition stages, predictions and likelihood analysis of diseaseprogression, particularly disease recurrence, metastatic spread ordisease relapse. A phenotype can also be a clinically distinct type orsubtype of a condition or disease, such as a cancer or tumor. Phenotypedetermination can also be a determination of a physiological condition,or an assessment of organ distress or organ rejection, such aspost-transplantation. The products and processes described herein allowassessment of a subject on an individual basis, which can providebenefits of more efficient and economical decisions in treatment.

The phenotype can be any phenotype listed in U.S. Pat. No. 7,897,356.The phenotype can be a tumor, neoplasm, or cancer. A cancer detected orassessed by products or processes described herein includes, but is notlimited to, breast cancer, ovarian cancer, lung cancer, colon cancer,hyperplastic polyp, adenoma, colorectal cancer, high grade dysplasia,low grade dysplasia, prostatic hyperplasia, prostate cancer, melanoma,pancreatic cancer, brain cancer (such as a glioblastoma), hematologicalmalignancy, hepatocellular carcinoma, cervical cancer, endometrialcancer, head and neck cancer, esophageal cancer, gastrointestinalstromal tumor (GIST), renal cell carcinoma (RCC) or gastric cancer. Thecolorectal cancer can be CRC Dukes B or Dukes C-D. The hematologicalmalignancy can be B-Cell Chronic Lymphocytic Leukemia, B-CellLymphoma-DLBCL, B-Cell Lymphoma-DLBCL-germinal center-like, B-CellLymphoma-DLBCL-activated B-cell-like, and Burkitt's lymphoma. Thephenotype may also be a premalignant condition, such as Barrett'sEsophagus.

The phenotype can also be an inflammatory disease, immune disease, orautoimmune disease. For example, the disease may be inflammatory boweldisease (IBD), Crohn's disease (CD), ulcerative colitis (UC), pelvicinflammation, vasculitis, psoriasis, diabetes, autoimmune hepatitis,Multiple Sclerosis, Myasthenia Gravis, Type I diabetes, RheumatoidArthritis, Psoriasis, Systemic Lupus Erythematosis (SLE), Hashimoto'sThyroiditis, Grave's disease, Ankylosing Spondylitis Sjogrens Disease,CREST syndrome, Sclerodenna, Rheumatic Disease, organ rejection, graftversus host disease, Primary Sclerosing Cholangitis, or sepsis.

The phenotype can also be a cardiovascular disease, such asatherosclerosis, congestive heart failure, vulnerable plaque, stroke, orischemia. The cardiovascular disease or condition can be high bloodpressure, stenosis, vessel occlusion or a thrombotic event.

The phenotype can also be a neurological disease, such as MultipleSclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD),schizophrenia, bipolar disorder, depression, autism, Prion Disease,Pick's disease, dementia, Huntington disease (HD), Down's syndrome,cerebrovascular disease, Rasmussen's encephalitis, viral meningitis,neurospsychiatric systemic lupus erythematosus (NPSLE), amyotrophiclateral sclerosis, Creutzfeldt-Jacob disease,Gerstmann-Straussler-Scheinker disease, transmissible spongiformencephalopathy, ischemic reperfusion damage (e.g. stroke), brain trauma,microbial infection, or chronic fatigue syndrome. The phenotype may alsobe a condition such as fibromyalgia, chronic neuropathic pain, orperipheral neuropathic pain.

The phenotype may also be an infectious disease, such as a bacterial,viral or yeast infection.

For example, the disease or condition may be Whipple's Disease, PrionDisease, cirrhosis, methicillin-resistant Staphylococcus aureus, HIV,hepatitis, syphilis, meningitis, malaria, tuberculosis, or influenza.Viral proteins, such as HIV or HCV-like particles can be assessed in anexosome, to characterize a viral condition.

The phenotype can also be a perinatal or pregnancy related condition(e.g. preeclampsia or preterm birth), metabolic disease or condition,such as a metabolic disease or condition associated with ironmetabolism. The metabolic disease or condition can also be diabetes,inflammation, or a perinatal condition.

The phenotype may be detected via any suitable assay method, such as,for example, western blots, ELISA, PCR, and the like. The assay methodsmay be combined to perform multiplexed analysis of more than onephenotype. Examples of assay methods that may be applied to themicrovesicles of the present invention are disclosed in PCT ApplicationsWO2009092386A3 and WO2012108842A1.

In the case where the biomarker is RNA, the RNA may be isolated from themicrovesicles of the present invention by the methods disclosed in U.S.Pat. No. 8,021,847.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for the diseases disclosed in U.S. Pat.No. 7,897,356.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for cancer according to the methodsdisclosed in U.S. Pat. No. 8,211,653.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for cancer according to the methodsdisclosed in U.S. Pat. No. 8,216,784.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for prostate cancer according to themethods disclosed in U.S. Pat. No. 8,278,059.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for the prognosis for cancer survivalaccording to the methods disclosed in U.S. Pat. No. 8,343,725.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for the prognosis for cancer survivalaccording to the methods disclosed in U.S. Pat. No. 8,349,568.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for acute lymphomic leukemia according tothe methods disclosed in U.S. Pat. No. 8,349,560.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for acute lymphomic leukemia according tothe methods disclosed in U.S. Pat. No. 8,349,561.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for hepatitis C virus. In one embodiment,hepatitis C viral RNA is extracted from the microvesicles of the presentinvention according to the methods described in U.S. Pat. No. 7,807,438to test for the presence of hepatitis C virus in a patient.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for determining the response of a patientto cancer therapy according to the methods disclosed in U.S. Pat. No.8,349,574.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for diagnosing malignant tumors accordingto the methods disclosed in U.S. Patent Application US20120058492A1.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for diagnosing cancer or adverse pregnancyoutcome according to the methods disclosed in U.S. Patent ApplicationUS20120238467A1.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for HIV in urine according to the methodsdisclosed in U.S. Patent Application US20120214151A1.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for cardiovascular events according to themethods disclosed in U.S. Patent Application US20120309041A1.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for cardiovascular events according to themethods disclosed in PCT Application WO2012110099A1.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for cardiovascular events according to themethods disclosed in PCT Application WO2012126531A1.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for cardiovascular events according to themethods disclosed in PCT Application WO2013110253A3.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for melanoma according to the methodsdisclosed in PCT Application WO2012135844A2.

In one embodiment, the microvesicles of the present invention areutilized in a diagnostic test for metastatic melanoma by testingmicrovesicles isolated according to the methods of the present inventionfor the presence of the biomarker BRAF. The presence of BRAF may bedetermined via western blot, or, alternatively, by PCR. In oneembodiment, the metastatic melanoma test is capable of detecting wildtype and malignant BRAF. In one embodiment, the metastatic melanoma testis capable of detecting splice variants of the malignant BRAF.

In one embodiment, the microvesicles that are utilized in the diagnostictest for metastatic melanoma are isolated using a method comprising thesteps outlined in FIG. 3 .

In one embodiment, microvesicles are obtained from a patient wishing tobe diagnosed for the presence of metastatic melanoma. In one embodiment,the microvesicles are obtained from the patient's plasma.

In one embodiment, the presence of metastatic melanoma is determined viaPCR, using one of the two primer sets below:

Sequence 1: Forward: AGACCTCACAGTAAAAATAGGTGAReverse: CTGATGGGACCCACTCCATC Amplicon length: 70 Sequence 2:Forward: GAAGACCTCACAGTAAAAATAGGTG Reverse: CTGATGGGACCCACTCCATCAmplicon length: 82

In another embodiment, the presence of metastatic melanoma is determinedvia western blot, using the mouse anti-BRAF V600E antibody (NewEastBiosciences, Malvern, Pa.).

Use of the Microvesicles of the Present Invention in Therapies

The microvesicles of the present invention can be used as a therapy totreat a disease.

In one embodiment, the microvesicles of the present invention are usedas vaccines according to the methods described in U.S. PatentApplication US20030198642A1.

In one embodiment, the microvesicles of the present invention are usedto modulate or suppress a patient's immune response according to themethods described in U.S. Patent Application US20060116321A1.

In one embodiment, the microvesicles of the present invention are usedto modulate or suppress a patient's immune response according to themethods described in PCT Patent Application WO06007529A3.

In one embodiment, the microvesicles of the present invention are usedto modulate or suppress a patient's immune response according to themethods described in PCT Patent Application WO2007103572A3.

In one embodiment, the microvesicles of the present invention are usedto modulate or suppress a patient's immune response according to themethods described in U.S. Pat. No. 8,288,172.

In one embodiment, the microvesicles of the present invention are usedas a therapy for cancer according to the methods described in PCT PatentApplication WO2011000551A1. In one embodiment, the microvesicles of thepresent invention are used as a therapy for cancer or an inflammatorydisease according to the methods described in U.S. Patent ApplicationUS20120315324A1.

In one embodiment, the microvesicles of the present invention are usedas a therapy for vascular injury according to the methods described inU.S. Pat. No. 8,343,485.

In one embodiment, the microvesicles of the present invention are usedto deliver molecules to cells. The delivery of molecules may be usefulin treating or preventing a disease. In one embodiment, the delivery isaccording to the methods described in PCT Application WO04014954A1. Inan alternate embodiment, the delivery is according to the methodsdescribed in PCT Application WO2007126386A1. In an alternate embodiment,the delivery is according to the methods described in PCT ApplicationWO2009115561A1. In an alternate embodiment, the delivery is according tothe methods described in PCT Application WO2010119256A1.

In one embodiment, the microvesicles of the present invention are usedto promote or enhance wound healing. In one embodiment, the wound is afull-thickness burn. In one embodiment, the wound is a second-degreeburn.

In one embodiment, the microvesicles of the present invention are usedto promote or enhance angiogenesis in a patient.

In one embodiment, the microvesicles of the present invention are usedto promote or enhance neuronal regeneration in a patient.

In one embodiment, the microvesicles of the present invention are usedto reduce scar formation in a patient.

In one embodiment, the microvesicles of the present invention are usedto reduce wrinkle formation in the skin of a patient.

In one embodiment, the microvesicles of the present invention are usedto orchestrate complex tissue regeneration in a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that can promote functional regenerationand organization of complex tissue structures. In one embodiment thepresent invention provides an isolated preparation of microvesicles thatcan regenerate hematopoietic tissue in a patient with aplastic anemia.In one embodiment the present invention provides an isolated preparationof microvesicles that can regenerate at least one tissue in a patientwith diseased, damages or missing skin selected from the groupconsisting of: epithelial tissue, stromal tissue, nerve tissue, vasculartissue and adnexal structures. In one embodiment, the present inventionprovides an isolated preparation of microvesicles that can regeneratetissue and/or cells from all three germ layers.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that is used to modulate the immune systemof a patient.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles that enhances the survival of tissue orcells that is transplanted into a patient. In one embodiment, thepatient is treated with the isolated preparation of microvesicles priorto receiving the transplanted tissue or cells. In an alternateembodiment, the patient is treated with the isolated preparation ofmicrovesicles after receiving the transplanted tissue or cells.

In an alternate embodiment, the tissue or cells is treated with theisolated preparation of microvesicles. In one embodiment, the tissue orcells is treated with the isolated preparation of microvesicles prior totransplantation.

In one embodiment, the present invention provides an isolatedpreparation of microvesicles containing at least one molecule selectedfrom the group consisting of RNA, DNA, lipid, carbohydrate, metabolite,protein, and combination thereof from a host cell. In one embodiment,the host cell is engineered to express at least one molecule selectedfrom the group consisting of RNA, DNA, lipid, carbohydrate, metabolite,protein, and combination thereof. In one embodiment, the isolatedpreparation of microvesicles containing at least one molecule selectedfrom the group consisting of RNA, DNA, lipid, carbohydrate, metabolite,protein, and combination thereof from a host cell is used as atherapeutic agent.

The present invention is further illustrated, but not limited by, thefollowing examples.

EXAMPLES Example 1: Isolation of Microvesicles from Cell Culture Mediumby Ultracentrifugation

This example illustrates the typical method by which microvesicles areisolated from cell culture medium, or any biological fluid. An outlineof the method to isolate microvesicles from cell culture medium is shownin FIG. 1 . In summary, the cells are cultured in medium supplementedwith microvesicle-free serum (the serum may be depleted of microvesiclesby ultracentrifugation, filtration, precipitation, etc.). Afterculturing the cells for a period of time, the medium is removed andtransferred to conical tubes and centrifuged at 400×g for 10 minutes at4° C. to pellet the cells. Next, the supernatant is transferred to newconical tubes and centrifuged at 2000×g for 30 minutes at 4° C. tofurther remove cells and cell debris. This may be followed by anothercentrifugation step (e.g. 10000×g for 30 minutes to further depletecellular debris and/or remove larger microvesicles). The resultantsupernatant is transferred to ultracentrifuge tubes, weighed to ensureequal weight and ultracentrifuged at 70000+×g for 70 minutes at 4° C. topellet the microvesicles.

This supernatant is subsequently discarded and the pellet is resuspendedin ice cold PBS. The solution is ultracentrifuged at 70000+×g for 70minutes at 4° C. to pellet the microvesicles. The microvesicle enrichedpellet is resuspended in a small volume (approximately 50-100 μl) of anappropriate buffer (e.g. PBS).

Example 2: Isolation of Microvesicles from Cell Culture Medium by theMethods of the Present Invention

This example illustrates how microvesicles are isolated from cellculture medium by the methods of the present invention. An outline ofthe method to isolate microvesicles from medium that has cultured cellsis shown in FIGS. 2 and 3 . In summary, the cells are cultured in mediumsupplemented with microvesicle-free serum (the serum may be depleted ofmicrovesicles by ultracentrifugation, filtration, precipitation, etc.).After culturing the cells for a period of time, the medium is removedand transferred to conical tubes and centrifuged at 400×g for 10 minutesat 4° C. to pellet the cells. Next, the supernatant is transferred tonew conical tubes and centrifuged at 2000×g for 30 minutes at 4° C. tofurther remove cells and cell debris. This may be followed by anothercentrifugation step (e.g. 10000×g for 30 minutes to further depletecellular debris and remove larger particles).

Microvesicles are then precipitated at 4° C. using 8.5% w/v PEG 6000 and0.4 M NaCl. This mixture is spun at 10000×g at 4° C. for 30 minutes. Thesupernatant is removed and the pellet is resuspended in an appropriatebuffer (e.g. PBS). It may be used for immediate downstream reactions orfurther purified. Further purification procedures can include the use ofcentrifugal filters (e.g. MWCO of 100 kDa), immunoaffinity, HPLC,tangential flow filtration, phase separation/partitioning,microfluidics, etc.

Example 3: Isolation of Microvesicles from Culture Medium ConditionedUsing Bone Marrow Derived Stem Cells by the Methods of the PresentInvention

Normal donor human bone marrow was acquired from AllCells LLC(Emeryville, Calif., http://www.allcells.com). MSCs were isolated by astandard plastic adherence method. Bone marrow mononuclear cells wereisolated by low-density centrifugation using Ficoll-Paque Premium(density: 1.077 g/ml) according to the manufacturer's protocol (GEHealthcare Life Sciences, Pittsburgh, Pa.). The mononuclear cells werecollected at the interface, washed three times in phosphate-bufferedsaline (PBS) supplemented with 2% FBS (Atlanta Biologics, Atlanta, Ga.),and resuspended in MSC medium consisting of alpha-minimum essentialmedium (α-MEM) (Mediatech Inc., Manassas, Va.) and 20% FBS, 1%Penicillin/Streptomycin (Lonza, Allendale, N.J.) and 1% glutamine(Lonza).

Initial cultures of either MSC's or mononuclear cells were seededbetween 2-3×10⁵ cells/cm² in tissue culture-treated dishes (BDBiosciences, San Jose, Calif.) and placed in a cell incubator at 37° C.in 95% humidified air and 5% CO₂. After 48-72 hours, the non-adherentcells were removed, the culture flasks were rinsed once with PBS, andfresh medium was added to the flask. The cells were grown until 80%confluence was reached and then passaged by Trypsin-EDTA (Lifetechnologies, Carlsbad, Calif.). Cells were split at a 1:4 ratio into5-layer multiflasks (BD Biosciences). Alternatively, cryopreserved MSCwere thawed at 37° C. and immediately cultured in α-MEM supplementedwith 20% microvesicle-free fetal bovine serum and 1%penicillin/streptomycin/glutamine at 37° C. in 95% humidified air and 5%CO₂. They were expanded similar to above.

The cells were grown in the multiflasks until 80-90% confluence wasreached. The flasks were rinsed twice with PBS and α-MEM supplementedwith 1% Penicillin/Streptomycin/Glutamine was added. After 24 hours, theconditioned medium transferred to 50 mL conical centrifuge tubes (ThermoFisher Scientific Inc, Weston, Fla.) and immediately centrifuged at400×g for 10 minutes at 4° C. to pellet any non-adherent cells. Thesupernatant was transferred to new 50 mL conical centrifuge tubes andcentrifuged at 2000×g for 30 minutes at 4° C. to further remove cellsand cell debris.

The supernatants were collected and placed into 250 ml sterile,polypropylene disposable containers (Corning, Corning, N.Y.). To thesupernatant, rnase and protease free polyethylene glycol averagemolecular weight 6000 (Sigma Aldrich, Saint Louis, Mo.) at 8.5 w/v % andsodium chloride (final concentration 0.4 M) were added. The solution wasplaced in a cold room at 4° C. overnight with rocking. The solution wastransferred to 50 mL conical centrifuge tubes and centrifuged at 10000×gat 4° C. for 30 minutes. The supernatant was decanted and themicrovesicle enriched pellet resuspended in phosphate-buffered saline(PBS). The microvesicle enriched solution was transferred to amiconultra-15 centrifugal filter units (nominal molecular weight limit 100kDa) (Millipore, Billerica, Mass.) and centrifuged at 5000×g for 30minutes. The filter units were washed with phosphate-buffered saline andcentrifuged again at 5000×g for 30 minutes. The concentrated sample wasrecovered (approximately 200 μl) from the bottom of the filter device.Protein concentration was determined by the micro BSA Protein assay kit(Pierce, Rockford, Ill.) and the enriched microvesicle solution wasstored at −70 degrees or processed for downstream use (e.g. protein,RNA, and DNA extraction).

Example 4: Isolation of Microvesicles from Plasma by the Methods of thePresent Invention

Approximately 6-8 ml of blood (human and pig) was collected viavenipuncture and placed into BD Vacutainer plastic EDTA lavender tubes(BD Biosciences, San Jose, Calif.). The venipuncture tubes werecentrifuged at 400×g for 30 minutes at room temperature. Plasma wasremoved (approximately 3-4 ml) and placed into new 50 ml conicalcentrifuge tubes (Thermo Fisher Scientific Inc, Weston, Fla.). Sterilealpha-minimum essential medium (α-MEM) (Mediatech Inc., Manassas, Va.)was added in a 1:10 (Plasma to medium) ratio.

To the solution, rnase and protease free polyethylene glycol averagemolecular weight 6000 (Sigma Aldrich, Saint Louis, Mo.) at 8.5 w/v % andsodium chloride (final concentration 0.4 M) were added. The solution wasplaced in a cold room at 4° C. overnight with rocking. The solution wascentrifuged at 10000×g at 4° C. for 30 minutes. The supernatant wasdecanted and the microvesicle enriched pellet resuspended inphosphate-buffered saline (PBS). The microvesicle enriched solution wastransferred to amicon ultra-15 centrifugal filter units (nominalmolecular weight limit 100 kDa) (Millipore, Billerica, Mass.) andcentrifuged at 5000×g for 30 minutes. The filter units were washed withphosphate-buffered saline and centrifuged again at 5000×g for 30minutes. The concentrated sample was recovered (approximately 200-400μl) from the bottom of the filter device. Protein concentration wasdetermined by the micro BSA Protein assay kit (Pierce, Rockford, Ill.)and the enriched microvesicle solution was stored at −70 degrees orprocessed for downstream use (e.g. protein, RNA, and DNA extraction).

Example 5: Isolation of Microvesicles from Bone Marrow Aspirate by theMethods of the Present Invention

Pig bone marrow was isolated from the iliac crest. The skin area wascarefully cleaned with povidine iodine 7.5% and isopropanol 70%. An11-gauge 3 mm trocar (Ranafac, Avon, Mass.) was inserted into the iliaccrest. An aspiration syringe with loaded with 5000-1000 units of heparinto prevent clotting of the marrow sample. Approximately 20-25 ml ofmarrow was aspirated and the solution transferred to 50 ml conicalcentrifuge tubes. Alternatively, normal donor human bone marrow(approximately 50 ml) was acquired from AllCells LLC (Emeryville,Calif., http://www.allcells.com).

The 50 ml conical tubes were centrifuged at 400×g for 30 minutes at roomtemperature. The supernatant (the acellular portion) was collected(approximately 10-12 ml per 50 ml) and placed into new 50 ml conicalcentrifuge tubes (Thermo Fisher Scientific Inc, Weston, Fla.). Sterilealpha-minimum essential medium (α-MEM) (Mediatech Inc., Manassas, Va.)was added in a 1:10 (bone marrow supernatant to medium) ratio. Thesolution was transferred to new 50 ml conical tubes and centrifuged at2000×g for 30 minutes at 4° C. The supernatant was transferred to new 50ml conical tubes and to this solution, rnase and protease freepolyethylene glycol average molecular weight 6000 (Sigma Aldrich, SaintLouis, Mo.) at 8.5 w/v % and sodium chloride (final concentration 0.4 M)were added.

The solution was placed in a cold room at 4° C. overnight with rocking.The solution was centrifuged at 10000×g at 4° C. for 30 minutes. Thesupernatant was decanted and the microvesicle enriched pelletresuspended in phosphate-buffered saline (PBS). The microvesicleenriched solution was transferred to amicon ultra-15 centrifugal filterunits (nominal molecular weight limit 100 kDa) (Millipore, Billerica,Mass.) and centrifuged at 5000×g for 30 minutes. The filter units werewashed with phosphate-buffered saline and centrifuged again at 5000×gfor 30 minutes. The concentrated sample was recovered (approximately200-400 μl) from the bottom of the filter device. Protein concentrationwas determined by the micro BSA Protein assay kit (Pierce, Rockford,Ill.) and the enriched microvesicle solution was stored at −70 degreesor processed for downstream use (e.g. protein, RNA, and DNA extraction).

The cellular portion was collected and processed for mesenchymal stemisolation or for bone marrow complete isolation.

Example 6: Isolation of Microvesicles from Urine by the Methods of thePresent Invention

Approximately 500 ml of clean catch human urine was isolated and placedinto 50 ml conical tubes (Thermo Fisher Scientific Inc, Weston, Fla.).

The 50 ml conical tubes were centrifuged at 400×g for 30 minutes at 4°C. The supernatant was removed and placed into new 50 ml conicalcentrifuge tubes (Thermo Fisher Scientific Inc, Weston, Fla.). Thesolution was transferred to new 50 ml conical tubes and centrifuged at2000×g for 30 minutes at 4° C. The supernatant was transferred to new 50ml conical tubes and to this solution, rnase and protease freepolyethylene glycol average molecular weight 6000 (Sigma Aldrich, SaintLouis, Mo.) at 8.5 w/v % and sodium chloride (final concentration 0.4 M)were added.

The solution was placed in a cold room at 4° C. overnight with rocking.The solution was centrifuged at 10000×g at 4° C. for 30 minutes. Thesupernatant was decanted and the microvesicle enriched pelletresuspended in phosphate-buffered saline (PBS). The microvesicleenriched solution was transferred to amicon ultra-15 centrifugal filterunits (nominal molecular weight limit 100 kDa) (Millipore, Billerica,Mass.) and centrifuged at 5000×g for 30 minutes. The filter units werewashed with phosphate-buffered saline and centrifuged again at 5000×gfor 30 minutes. The concentrated sample was recovered (approximately200-400 μl) from the bottom of the filter device. Protein concentrationwas determined by the micro BSA Protein assay kit (Pierce, Rockford,Ill.) and the enriched microvesicle solution was stored at −70 degreesor processed for downstream use (e.g. protein, RNA, and DNA extraction).

Example 7: Isolation of Microvesicles from Medium from a Long-TermCulture of Bone Marrow Cells by the Methods of the Present Invention

Bone marrow was obtained from an aspirate (see example 1) and red bloodcells were lysed using 0.8% ammonium chloride solution containing 0.1 mMEDTA (Stem Cell Technologies, Vancouver, BC). The nucleated cells werepelleted under a fetal bovine serum (Atlanta Biologics, Atlanta, Ga.)cushion at 400×g for 5 minutes. Nucleated cells were washed in McCoys 5amedia (Mediatech Inc., Manassas, Va.) by pelleting at 400×g for 5 min.The cells were resuspended in culture media at a density of 1×10⁶cells/ml and plated in 25, 75 or 225 cm² flasks (Corning, Corning,N.Y.).

Culture media consisted of McCoy's 5a media, 1% sodium bicarbonate (Lifetechnologies, Carlsbad, Calif.), 0.4% MEM non-essential amino acids(Life technologies), 0.8% MEM essential amino acids (Life technologies),1% L-glutamine (Lonza, Allendale, N.J.), 0.1 μM Hydrocortisone (Lifetechnologies), 1% penicillin/streptomycin (Lonza), 12-5% fetal calfserum (Atlanta Biologics) and 12-5% horse serum (Stem Cell Technology).The cultures were incubated at 33° C. and 5% CO₂. Feeding was performedweekly by adding half of the original volume of media without removingany media during the first nine weeks of culture. If the cultures weregrown beyond nine weeks, the volume of culture media was reduced to theoriginal volume and half the original volume of fresh media was addedeach week.

After approximately nine weeks of culture, the original medium wasremoved and stored. The cells were washed twice with phosphate bufferedsaline (PBS) and incubated for 24 hours in media consisting of McCoy's5a media, 1% sodium bicarbonate, 0.4% MEM non-essential amino acids,0.8% MEM essential amino acids (Life technologies), 1% L-glutamine(Lonza, Allendale, N.J.), and 1% penicillin/streptomycin (Lonza).

After 24 hours, the supernatant was transferred to 50 mL conicalcentrifuge tubes (Thermo Fisher Scientific Inc, Weston, Fla.) andimmediately centrifuged at 400×g for 10 minutes at 4° C. to pellet anynon-adherent cells. The original medium that was stored was added backto the cells. The supernatant were transferred to new 50 mL conicalcentrifuge tubes and centrifuged at 2000×g for 30 minutes at 4° C. tofurther remove cells and cell debris.

The supernatant was collected and placed into 250 ml sterile,polypropylene disposable containers (Corning, Corning, N.Y.). To thesupernatant, rnase and protease free polyethylene glycol averagemolecular weight 6000 (Sigma Aldrich, Saint Louis, Mo.) at 8.5 w/v % andsodium chloride (final concentration 0.4 M) was added. The solution wasplaced in a cold room at 4° C. overnight with rocking. The solution wastransferred to 50 mL conical centrifuge tubes and centrifuged at 10000×gat 4° C. for 30 minutes. The supernatant was decanted and themicrovesicle enriched pellet resuspended in phosphate-buffered saline(PBS). The microvesicle enriched solution was transferred to amiconultra-15 centrifugal filter units (nominal molecular weight limit 100kDa) (Millipore, Billerica, Mass.) and centrifuged at 5000×g for 30minutes. The filter units were washed with phosphate-buffered saline andcentrifuged again at 5000×g for 30 minutes. The concentrated sample wasrecovered (approximately 200 μl) from the bottom of the filter device.Protein concentration was determined by the micro BSA Protein assay kit(Pierce, Rockford, Ill.) and the enriched microvesicle solution storedat −70 degrees or processed for downstream use (e.g. protein, RNA, andDNA extraction).

Example 8: Analysis of the Microvesicles of the Present Invention

Samples of microvesicles were analyzed by electron microscopy. Fortransmission electron microscopy (TEM), each specimen of microvesicleswas loaded on formvar-coated, 150 mesh copper grids (Electron MicroscopySciences, Fort Washington, Pa.) for 20 minutes. The grids were drainedand floated on drops of 2% glutaraldehyde for 5 minutes, then washed indouble distilled water (DDOH), followed by staining on drops of 4%aqueous uranyl acetate and multiple washes in DDOH. The grids wereexamined at 80 kV in a Philips CM10 electron microscope.

FIG. 5 shows electron micrographs of microvesicles derived from humanbone marrow-derived mesenchymal stem cells isolated by theultracentrifuge method described in Example 1 (panels A&B) and accordingto the methods of the present invention as described in Example 3(panels C&D). FIG. 6 shows electron micrographs of microvesicles derivedfrom porcine bone marrow-derived mesenchymal stem cells isolated by theultracentrifuge method described in Examples 1 (panels A&B) andaccording to the methods of the present invention as described inExample 3 (panels C&D). FIG. 7 shows electron micrographs ofmicrovesicles derived from murine bone marrow-derived mesenchymal stemcells isolated by the ultracentrifuge method described in Examples 1(panels A&B) and according to the methods of the present invention asdescribed in Example 3 (panels C&D).

FIGS. 5 to 7 illustrate the differences between microvesicles isolatedby the methods of the present invention compared to ultracentrifugeisolation. The microvesicles isolated according to the methods of thepresent invention have borders that are smoother, uncorrugated andappear more “intact.”

FIG. 8 shows electron micrographs of microvesicles isolated from humanplasma according to the methods of the present invention. Theheterogeneity of the shapes and sizes achieved with PEG isolationsuggests that all types of microvesicles were isolated. Similarheterogeneity was observed in microvesicles from porcine plasma (FIG. 9) and human urine (FIG. 10 ) that were isolated according to the methodsof the present invention.

To analyze protein expression in samples of microvesicles, cells andmicrovesicles were lysed in RIPA buffer (Cell signaling technology,Danvers, Mass.) and protein concentration estimated by the microBSAassay kit (Pierce, Rockford, Ill.). Approximately 20 micrograms oflysate were loaded in each lane and the membranes were probed overnight(1:1000) by either Rabbit anti-63 antibody (SBI Biosciences, MountainView, Calif.), Rabbit anti-hsp70 (SBI Biosciences), rabbit STAT3 (Cellsignaling technology), and/or rabbit phospho-STAT3 (Cell signalingtechnology).

The presence of the exosomal markers (HSP 70 and CD63) confirmed thatthe methods of the present invention were capable of isolating exosomes.Further, the exosomes also contained the transcription factor STAT3 andthe activated phosphorylated form phospho-STAT3. See FIG. 11 .

Example 9: The Effect of the Microvesicles of the Present Invention onFibroblast Proliferation and Migration

To study the ability the microvesicles of the present invention topromote or enhance wound healing, the ability of the microvesicles tostimulate the proliferation of dermal fibroblasts was tested. Normalhuman adult dermal fibroblasts were obtained from Life Technology(Carlsbad, Calif.). Chronic wound patient fibroblasts (pressure footulcer and diabetic foot ulcer) were collected under an IRB approvedprotocol (IND # BB IND 13201) from wounds of 2 years duration withoutevidence of healing despite standard of care and advanced wound caretreatments. Normal and Chronic wound fibroblasts were plated at5×10{circumflex over ( )}3 cells per well on 24 well tissue cultureplates (BD Biosciences, San Jose, Calif.). MTT cell proliferation assayswere performed at day 0 and day 3. Microvescicles were added on day 0.Both PEG isolated and ultracentrifuge isolated microvesicles wereapproximately equivalent in increasing growth of both normal and chronicwound fibroblasts after 3 days. Phosphate buffered saline (PBS) andconditioned MSC medium depleted of microvesicles showed little growth.See FIG. 12 .

In coculture experiments, normal adult fibroblasts and fibroblast from adiabetic foot ulcer were seeded in twenty four well plates. Each wellwas seeded to achieve 100% confluency (approximately 1×10⁵ cells perwell). To prevent the influence of cell proliferation, 2 hours prior toscratch, the medium was substituted with a fresh serum-free culturemedium containing mitomycin at 10 μg/ml. The confluent monolayer wasthen scored with a 1 ml sterile pipette tip to leave a scratch of0.4-0.5 mm in width. Culture medium was then immediately removed (alongwith any dislodged cells). The removed medium was replaced with freshculture medium (10% FBS) containing either microvesicles (PEG orultracentrifuge derived), PBS, or MSC conditioned medium depleted ofmicrovesicles. The scratched area was monitored by collecting digitizedimages immediately after the scratch and 3 days after treatment.Digitized images were captured with an inverted IX81 Olympus microscope(Olympus America, Center Valley, Pa., http://www.olympusamerica.com) andORCA-AG Hamamatsu digital camera (Hamamatsu Photonics K.K., HamamatsuCity, Shizuoka Pref., Japan, http://www.hamamatsu.com). Three days aftertreatment, microvesicles isolated according to the methods of thepresent invention showed the greatest in migration (essentially closingthe wound), followed by microvesicles derived from ultracentrifuge. Thecontrols (PBS) and MSC conditioned medium depleted of microvesicles(Depleted) showed little migration. See FIG. 13 .

FIG. 14 shows the effects of microvesicles on cell migration fibroblastsderived from a diabetic foot ulcer. Similar to the results in FIG. 13 ,microvesicles isolated according to the methods of the present inventionevoked the greatest migration, followed by microvesicles isolated usingthe ultracentrifuge method described in Example 1. The controls (PBS)and MSC conditioned medium depleted of microvesicles (Depleted) showedlittle migration.

Example 10: Uptake of the Microvesicles of the Present Invention intoCells

Human MSC microvesicles isolated from conditioned medium according tothe methods of the present invention were labeled with the phospholipidcell linker dye PKH-26 (red) per manufacturer's instruction(Sigma-Aldrich, St. Louis, Mo.). Normal skin fibroblasts were labeledwith Vybrant-Dio (Life technology) per manufacturer instructions. Normalskin fibroblasts were plated on fibronectin (Sigma-Aldrich) coated4-well Nunc* Lab-Tek* II Chamber Slides (Thermo Fisher Scientific Inc,Weston, Fla.) (5×10³ cells per well). Cells were stained with thenuclear dye Hoechst 33342 (Life technology) per manufacturer'sinstructions. Dio labeled fibroblasts were treated with PKH-26 labeledmicrovesicles for 24 hours. Images were captured with an inverted IX81Olympus microscope and ORCA-AG Hamamatsu digital camera. Normal dermalfibroblasts (stained with the green lipid membrane dye Dio) demonstrateduptake of PKH-26 labeled human MSC MV isolated by PEG precipitation in aperi-nuclear location. See FIGS. 15 and 16 . In FIG. 16 , themicrovesicles are seen in a peri-nuclear location.

Example 11: Use of the Microvesicles of the Present Invention as aDiagnostic for Rheumatoid Arthritis

Normal dermal fibroblasts were plated at a density of 1×10⁵ cells/wellin a 6 well tissue culture plate (BD Biosciences). Fibroblasts wereserum starved overnight and treated with PBS (control), 10 micrograms ofeither microvesicles isolated according to the methods of the presentinvention from plasma obtained from a patient suffering from rheumatoidarthritis (Human Plasma MV PEG Precipitation); microvesicles isolatedaccording to the methods of the present invention from mediumconditioned with bone marrow-derived mesenchymal stem cells (Human hMSCMV PEG Precipitation); microvesicles isolated according viaultracentrifugation from medium conditioned with bone marrow-derivedmesenchymal stem cells (Human hMSC MV ultracentrifugation); PBS control;and a depleted medium control (hMSC conditioned medium depleted of MV).The amount of STAT3 phosphorlyation observed in the fibroblasts wasgreater in the microvesicles isolated according to the methods of thepresent invention. See FIG. 17 .

Example 12: Use of the Microvesicles of the Present Invention as aDiagnostic for Metastatic Melanoma

BRAF is a human gene that makes a protein called B-Raf. More than 30mutations of the BRAF gene associated with human cancers have beenidentified. We have designed pcr primers to amplify the mutated form ofBRAF that is linked to metastatic melanoma. The mutation is a T1799Amutation in exon 15 in BRAF. This leads to valine (V) being substitutedfor by glutamate (E) at codon 600 (now referred to as V600E). Thepresence of this mutation is required for treatment by the BRAFinhibitor Vemurafenib.

The SK-Mel28 cell line, obtained from ATCC (Washington D.C., Maryland)is known to have the T1799A mutation in exon 15 in BRAF. Microvesicles,isolated according to the methods of the present invention were obtainedfrom medium conditioned by a 3 day incubation in EMEM (ATCC)+10% serum(Atlanta biologics, Atlanta, Ga.).

The isolated microvesicles were processed for DNA and RNA isolationusing Qiagen's (Hilden, Germany) AllPrep DNA/RNA kit. Approximately 50ng of RNA from SK-MEL28 cells and microvesicles were reverse transcribedusing iScript™ Reverse Transcription Supermix (Biorad, Hercules,Calif.). A 2 μl aliquot was used for PCR utilizing Platinum® PCRSuperMix (Life technology) per manufacturer's instructions. In addition,80 ng of DNA from SK-MEL28 cells and microvesicles was used for PCRutilizing Platinum® PCR SuperMix per manufacturer's instructions. PCRproducts were run on a 3% agarose gel and visualized by Biorad's gel-docsystem. The results are shown in FIG. 18 .

The primers used were:

Sequence 1: Forward: AGACCTCACAGTAAAAATAGGTGAReverse: CTGATGGGACCCACTCCATC Amplicon length: 70 Sequence 2:Forward: GAAGACCTCACAGTAAAAATAGGTG Reverse: CTGATGGGACCCACTCCATCAmplicon length: 82

In addition, samples of the microvesicles were lysed in RIPA buffer andprotein concentration estimated by the microBSA assay kit. Approximately50 microgram were loaded in each lane and the membranes were probedovernight (1:1000) by mouse anti-BRAF V600E antibody (NewEastBiosciences, Malvern, Pa.). Secondary antibody, goat anti-mouse (Pierce)was applied at 1:10000 dilution for 1 hour. The Western blot shows BRAFV600E detection in SKMEL28 cell and MV lysate.

Example 13: Isolation of Microvesicles from Medium Conditioned Using aCulture of GFP-Labeled Bone Marrow-Derived Mesenchymal Stem Cells by theMethods of the Present Invention

Homozygous transgenic mice expressing the enhanced Green FluorescentProtein (GFP) under the direction of the human ubiqutin C promoter(C57BL/6-Tg(UBC-GFP)30Scha/J) were obtained from Jackson Laboratories(Bar Harbor, Me.). These mice are known to express GFP in all tissues.

GFP-Mice (approximately 3-4 weeks of age) were euthanized by CO₂asphyxiation. The limbs were cut above the hip and below the anklejoint. The hind limbs were harvested and skin, muscle, and allconnective tissue was removed. The bones were then placed in a dish ofice cold sterile 1×PBS and washed several times in PBS. The ends of eachbone were snipped off with scissors. A 10 cc syringe with warmed medium(α-MEM supplemented with 20% fetal bovine serum and 1%penicillin/streptomycin/glutamine) was forced through the bone shaft toextract all bone marrow into a 150 mm plate. This was repeated severaltimes to ensure all the marrow was removed. The cell mixture waspipetted several times to dissociate cells and the cell suspension waspassed through a cell strainer (70 μm size) (BD Biosciences, San Jose,Calif.) to remove large cell clumps or bone particles.

Initial cultures were seeded between 2-3×10⁵ cells/cm² in tissueculture-treated dishes (BD Biosciences, San Jose, Calif.) and placed ina cell incubator at 37° C. in 95% humidified air and 5% CO₂. After 72-96hours the non-adherent cells were removed, the culture flasks wererinsed once with PBS, and fresh medium was added to the flask. The cellswere grown until 80% confluence was reached and then passaged byTrypsin-EDTA (Life technologies, Carlsbad, Calif.). Cells were split ata 1:4 ratio.

Alternatively, cryopreserved GFP Mouse-MSC's were thawed at 37° C. andimmediately cultured in α-MEM supplemented with 20% fetal bovine serumand 1% penicillin/streptomycin/glutamine at 37° C. in 95% humidified airand 5% CO₂. They were expanded similar to above.

The cells were grown in the flasks until 100% confluence was reached(approximately 1 week). The supernatant were transferred to 50 mLconical centrifuge tubes (Thermo Fisher Scientific Inc, Weston, Fla.)and immediately centrifuged at 400×g for 10 minutes at 4° C. to pelletany non-adherent cells. The supernatant was transferred to new 50 mLconical centrifuge tubes and centrifuged at 2000×g for 30 minutes at 4°C. to further remove cells and cell debris.

The supernatants were collected and placed into 250 ml sterile,polypropylene disposable containers (Corning, Corning, N.Y.). To thesupernatant, rnase and protease free polyethylene glycol averagemolecular weight 6000 (Sigma Aldrich, Saint Louis, Mo.) at 8.5 w/v % andsodium chloride (final concentration 0.4 M) were added. The solution wasplaced in a cold room at 4° C. overnight with rocking. The solution wastransferred to 50 mL conical centrifuge tubes and centrifuged at 10000×gat 4° C. for 30 minutes. The supernatant was decanted and themicrovesicle enriched pellet resuspended in phosphate-buffered saline(PBS). The microvesicle enriched solution was transferred to amiconultra-15 centrifugal filter units (nominal molecular weight limit 100kDa) (Millipore, Billerica, Mass.) and centrifuged at 5000×g for 30minutes. The filter units were washed with phosphate-buffered saline andcentrifuged again at 5000×g for 30 minutes. The concentrated sample wasrecovered (approximately 200-400 μl) from the bottom of the filterdevice. Protein concentration was determined by the micro BSA Proteinassay kit (Pierce, Rockford, Ill.) and the enriched microvesiclesolution was stored at −70 degrees or processed for downstream use (e.g.protein, RNA, and DNA extraction).

To determine cellular uptake of the microvesicles, normal human skinfibroblasts were labeled with Vybrant-Dio (Life technology) permanufacturer instructions. Normal skin fibroblasts were plated onfibronectin (Sigma-Aldrich) coated 4-well Nunc* Lab-Tek* II ChamberSlide (Thermo Fisher Scientific Inc) (5×10³ cells per well). Cells werestained with the nuclear dye Hoechst 33342 (Life technology) permanufacturer's instructions. Dil labeled fibroblasts were treated withmicrovesicles isolated from GFP expressing mouse MSC for 24 hours.Images were captured with an inverted IX81 Olympus microscope andORCA-AG Hamamatsu digital camera. See FIGS. 20 and 21 . Importantly,these images show that the microvesicles containing GFP were taken up bythe cells.

Example 14: Use of the Microvesicles of the Present Invention as aTherapy to Promote or Enhance Wound Healing

Full thickness wounds were created on the backs of pigs using a 10 mmpunch biopsy instrument. Microvesicles were isolated from culture mediumconditioned using autologous bone marrow-derived mesenchymal stem cells,either according to the methods described in Example 1 (the“conventional” ultracentrifugation method”), or by the methods describedin Example 3. 30 micrograms of microvesicles were administered to thewounds by local injection at the time of wounding and at Days 1 and 2.Controls were treated with saline or allowed to heal air exposed. After5 days, the animals were euthanized, and the wounds examined.

FIG. 22 shows the histology of the wounds 5 days post wounding. At 5days, wounds treated with microvesicles isolated according to themethods of the present invention (i.e., according to the methodsdescribed in Example 3) appeared smaller than saline controls, airexposed controls and wounds treated with microvesicles prepared byultracentrifugation. The wounds treated with microvesicles prepared byultracentrifugation showed an enhanced inflammatory response, comparedto those treated with microvesicles prepared according to the methods ofthe present invention and both controls.

In another study, second degree burn wounds were created on the backs ofpigs using a brass rod heated to 100° C. Microvesicles were isolatedfrom culture medium conditioned using autologous bone marrow-derivedmesenchymal stem cells, either according to the methods described inExample 1 (the “conventional” ultracentrifugation method”), or by themethods described in Example 3. 30 micrograms of microvesicles wereadministered to the wounds by local injection at the time of woundingand at days 1 and 2. Controls were treated with saline or allowed toheal air exposed.

Over the course of the experiment (up to 28 Days post burn injury)wounds treated with microvesicles prepared by ultracentrifugation weresignificantly more inflamed than those treated with microvesiclesprepared according to the methods of the present invention (i.e.,according to the methods described in Example 3). See FIG. 23 .Similarly, wounds treated with microvesicles prepared byultracentrifugation were significantly more inflamed than salinecontrols and air exposed controls. Burn wounds treated withmicrovesicles prepared according to the methods of the present inventiondid not appear significantly more inflamed than controls.

FIG. 23 illustrates the difference in inflammation at Day 7 postwounding between wounds treated with microvesicles prepared byultracentrifugation, microvesicles prepared according to the methods ofthe present invention and an air exposed control. Microscopically,abscess formation was seen in both full thickness and burn woundstreated with microvesicles prepared by ultracentrifugation. Theinflammation noted with microvesicles prepared by ultracentrifugation isthought to be due to damaged microvesicles, which can easily stimulatean inflammatory cascade. The microvesicles of the present invention mayalso confer additional benefits by including additional particles.

FIG. 24 shows a second degree porcine burn wound treated withmicrovesicles isolated by the methods of the present invention 28 daysafter burn injury. There is a significant remodeling of collagen, withthe appearance of ground substance. These findings are indicative ofdermal remodeling with collagen type III formation. There is also dermalepidermal induction resulting in a thickened epidermis that appears wellanchored to the dermis. These findings are not observed in scarformation and are more consistent with dermal regeneration. An epidermisforming over a scar is easily subject to reinjury due to the inabilityto anchor well to a scarred dermis.

FIG. 25 shows a second degree porcine burn wound treated with saline 28days after burn injury. There is minimal dermal regeneration with aflattened epidermis. The lack of significant rete ridge formation ishighly suggestive of an inadequately anchored epidermis. These findingsare much more indicative of scar formation with the risk of continuedinjury.

FIG. 26 shows a full thickness porcine wound treated with microvesiclesisolated according to the methods of the present invention 28 days afterinjury. There is ingrowth of a nerve (illustrated by the arrows) intothe remodeling dermis, likely stimulated by the application of themicrovesicles. The nerve grown is accompanied by an angiogenic response(circled areas). The nerve appears to be a developed structure and isnot due to simple axon sprouting. This is a unique finding and has neverbeen reported and was also not observed in control wounds or woundstreated with microvesicles prepared by ultracentrifugation. Theseobservations are highly indicative of complex tissue regeneration withthe ability to generate mature elements from all germ layers includingepidermis, stroma, vasculature and nervous tissue. These methods thenappear to be widely applicable to the treatment of numerous conditionsincluding traumatic, inflammatory, neoplastic and degenerative disordersof ectodermal, endodermal and mesodermal derived tissues.

FIG. 27 shows a full thickness porcine wound treated with microvesiclesisolated by the methods of the present invention 28 days after injury.This Figure illustrates the observations described in FIG. 26 at greatermagnification. In A) the nerve growth appears to be following a pathrelated to the angiogenic response. This finding is interesting as nervegrowth is well known to follow angiogenesis in embryologic development.Again, these findings are indicative of tissue regeneration. B) showsthe nerve at higher power. C) better illustrates the angiogenesisadjacent to the nerve growth.

Bone formation was seen in all treatment groups (control andmicrovesicle treated) in the porcine full thickness wound model. SeeFIG. 28 . Animals received a total of 1.44 mg microvesicles (halfprepared according to the methods of the present invention and half byultracentrifugation). There then appeared to be a systemic effectstimulating the formation of bone in all wounds. Bone formation tendedto occur more in more inflammatory wounds suggesting a synergisticeffect of local inflammatory mediators and the systemic effect ofmicrovesicles.

Example 15: Use of the Microvesicles of the Present Invention as aTherapy to Repopulate Bone Marrow and Regenerate Complex Structures

C57/CJ6 (GFP⁻) mice were lethally irradiated with two cycles of 400 cGygamma irradiation to ablate their host bone marrow progenitors. Afterirradiation, mice were treated in an approximately 2 cm² area with anablative fractional Erbium:YAG laser. After laser treatment, a plasticchamber was adhered to the skin, and bone marrow derived cells obtainedfrom a syngeneic GFP⁺ transgenic mouse were added to the chamber. TheGFP⁺ bone marrow cells included, freshly harvested total bone marrowcells, lineage negative selected bone marrow cells, mesenchymal stemcells and bone marrow complete cultured cells (as described in thisapplication). In only a few animals was chimerism able to be achieved;detected by circulating GFP⁺ cells 4 to 6 weeks after administration ofcells. See FIG. 29 . Surprisingly, many animals survived withoutevidence of donor bone marrow engraftment. Overall (in all groups ofcells given) 30% of animals receiving cells survived. Among thedifferent groups, survival rates were highest for animals receivinglineage negative selected cells (45%) and fresh bone marrow cells (30%).Control irradiated animals receiving no cells had a 100% mortality rate.Cytokines have failed to similarly rescue similarly lethally irradiatedanimals and no functional donor bone marrow engraftment could bedemonstrated in these surviving animals. Microvesicles secreted by thedelivered cells are likely responsible for the recovery of the host bonemarrow leading to survival of these animals. We have demonstrated thatfresh bone marrow (which includes lineage negative cells) andmesenchymal stem cells produce ample amounts of microvesicles that couldaccomplish this effect.

In another study, C57/CJ6 (GFP) mice were lethally irradiated with twocycles of 400 cGy gamma irradiation to inhibit their hair growth andpartially ablate their bone marrow. After irradiation, the backs of themice were shaved and the mice were then in an approximately 2 cm² areawith an ablative fractional Erbium:YAG laser. After laser treatment, aplastic chamber was adhered to the skin and bone marrow derived cellsobtained from a syngeneic GFP⁺ transgenic mouse were added to thechamber. The GFP⁺ bone marrow cells included, freshly harvested bonemarrow cells, lineage negative selected bone marrow cells, mesenchymalstem cells and bone marrow complete cultured cells (as described in thisapplication). In no animals was chimerism able to be achieved; detectedby circulating GFP⁺ cells 4 to 6 weeks after administration of cells.See FIG. 30 . Animals receiving laser treatment alone had no to veryminimal short stubby hair growth. FIG. 30 (A). In animals given bonemarrow cells, there was significant, long lasting hair growth. FIG. 30(A & B). These findings were most dramatic in mice treated with GFP⁺lineage negative selected cells and total fresh GFP⁺ bone marrow cells.Hair growth could be detected in 2 weeks and continued to grow forseveral months. Skin biopsies were taken in the area of new hair growthbut no GFP⁺ cells were detected. Functional engraftment of bone marrowcells could also not be detected in any animal by FACS analysis. FIG. 30(C). As with the example in FIG. 29 , cytokines have not beendemonstrated to have this effect in restoring hair growth. Microvesiclessecreted by the delivered cells are likely responsible for thestimulation of hair growth.

Example 16: Use of the Microvesicles of the Present Invention to Promoteor Stimulate Angiogenesis and to Promote or Stimulate FibroblastProliferation

Isolation of Bone Marrow Aspirate Microvesicles: Approximately 25 ml offresh whole bone marrow was obtained from Allcells, Inc. (Alameda,Calif.). The bone marrow was carefully placed into new 50 ml conicalcentrifuge tubes and centrifuged at 400×g for 30 minutes at roomtemperature. The supernatant was carefully removed (approximately 15 ml)and placed into new 50 ml conical centrifuge tubes (Thermo FisherScientific Inc, Weston, Fla.) and centrifuged at 2000×g for 30 minutesat 4° C. The supernatant was again carefully removed and placed into new50 ml conical centrifuge tubes to which sterile alpha-minimum essentialmedium (α-MEM) (Mediatech Inc., Manassas, Va.) was added in a 1:10 (Bonemarrow supernatant to medium) ratio. To the solution, rnase and proteasefree polyethylene glycol average molecular weight 6000 (Sigma Aldrich,Saint Louis, Mo.) at 8.5 w/v % and sodium chloride (final concentration0.4 M) were added. The solution was placed in a cold room at 4° C.overnight with rocking. The solution was centrifuged at 10000×g at 4° C.for 30 minutes. The supernatant was decanted and the microvesicleenriched pellet resuspended in phosphate-buffered saline (PBS). Themicrovesicle enriched solution was transferred to amicon ultra-15centrifugal filter units (nominal molecular weight limit 100 kDa)(Millipore, Billerica, Mass.) and centrifuged at 5000×g for 30 minutes.The filter units were washed with phosphate-buffered saline andcentrifuged again at 5000×g for 30 minutes. The concentrated sample wasrecovered (approximately 200-400 μl) from the bottom of the filterdevice.

Angiogenesis assay: Angiogenesis was measured using an endothelial tubeformation assay (Invitrogen Life Technologies, Grand Island, N.Y.).Cryopreserved primary Human Umbilical Vein Endothelial cells (HUVEC)(Invitrogen Life Technologies) were grown in a 75-cm² tissue-cultureflask for 6 days in Medium 200PRF supplemented with 2% low serum growthsupplement (Invitrogen Life Technologies). Cells were then plated at adensity of 3×10⁴ in a 24 well tissue culture plate containing mediumwithout supplement. HUVEC Cells were subsequently treated with bonemarrow microvescles (approximately 100 μg). PBS was used as the vehiclecontrol. Treated cells were incubated for 6 hours at 37° C. and 5% CO².Calcein AM fluorescent dye at a concentration of 2 μg/ml was used forvisualization of tube formation. Fluorescent images were captured withan inverted IX81 Olympus microscope (Olympus America, Center Valley,Pa.). Bone marrow MV showed significant tube formation capacity ascompared to the vehicle (PBS) control (see FIG. 31 ).

Growth assay: Normal adult fibroblasts were plated onto 24-well plates(10000 cells/well) in growth media (5% FBS, 1% glutamine, 1%Penicillin/Streptomycin) for the assays. After overnight incubation,three wells were randomly selected and the cells were stained withNucBlue Live ReadyProbes Reagent (Invitrogen Life technologies) (Day 0).Fluorescent images were captured using the EVOS FL Auto Cell ImagingSystem (Invitrogen Life technologies). Fibroblasts were re-fed withfresh medium containing bone marrow-derived microvesicles (approximately100 μg) or PBS (vehicle control) and after three days (Day 3), cellswere stained and imaged. Bone marrow-derived microvesicle treatedfibroblasts increased approximately three fold in number (compared toDay 0) and at a significant greater rate than the vehicle control (FIG.32 , panel A and FIG. 32 , panel B).

Although the various aspects of the invention have been illustratedabove by reference to examples and preferred embodiments, it will beappreciated that the scope of the invention is defined not by theforegoing description but by the following claims properly construedunder principles of patent law.

What is claimed is:
 1. A topical composition for treating wounds comprising an effective amount of an enriched population of intact microvesicles collected from polyethylene glycol precipitation of a mesenchymal stem cell supernatant or a biological fluid; wherein the mesenchymal stem cells are derived from bone marrow; wherein the microvesicles range in size from 2 nm to 5000 nm; wherein the topical composition further comprises a storage solution; wherein the intact microvesicles result in increased migration of non-proliferating cultured fibroblasts in a cell migration assay when administered to the non-proliferating cultured fibroblasts; and wherein said effective amount of isolated microvesicles results in regeneration of at least one skin tissue in the skin wound.
 2. The topical composition of claim 1, wherein the microvesicles are heterogeneous in size and shape and comprise ectosomes, exosomes, microparticles, microvesicles, nanovesicles, shedding vesicles, apoptotic bodies, and/or membrane particles.
 3. The topical composition of claim 1, wherein the microvesicles do not provoke a significant inflammatory response when administered to the wound a subject.
 4. The topical composition of claim 1, wherein the microvesicles range in size from 2 nm to 200 nm.
 5. The topical composition of claim 1, wherein the microvesicles have a molecular weight of at least 100 kDa.
 6. The topical composition of claim 1, wherein administration of the topical composition to the wound of a subject results in a reduction of wrinkle formation or scar formation in skin of the wound of the subject.
 7. The topical composition of claim 1, wherein the microvesicles express the exosomal markers HSP 70, CD63, and STAT3.
 8. The topical composition of claim 1, wherein the microvesicles contain at least one molecule selected from the group consisting of RNA, DNA, lipids, carbohydrates, drugs, small molecules, peptides, metabolites, and proteins from a host cell.
 9. The topical composition of claim 8, wherein the host cell is engineered to express the at least one RNA, DNA, peptide, or protein molecule.
 10. The topical composition of claim 1, wherein the composition is formulated for topical administration.
 11. A topical composition for treating wounds comprising an effective amount of an enriched population of intact microvesicles, comprising ectosomes, exosomes, microparticles, microvesicles, nanovesicles, shedding vesicles, apoptotic bodies, and/or membrane particles; wherein the topical composition further comprises a buffer or stabilization solution; wherein the microvesicles are isolated from polyethylene glycol precipitation of a bone marrow mesenchymal stem cell supernatant or a biological fluid; wherein the microvesicles comprise a size ranging from 2 nm to 5000 nm; wherein the intact microvesicles result in increased migration of non-proliferating cultured fibroblasts in a cell migration assay when administered to the non-proliferating cultured fibroblasts; and wherein said effective amount of isolated microvesicles results in regeneration of at least one skin tissue in the skin wound.
 12. The topical composition of claim 11, wherein the isolated microvesicles have a molecular weight of at least 100 kDa.
 13. The topical composition of claim 11, wherein the composition is formulated for topical administration. 